Methods and compositions for nucleic acid amplification

ABSTRACT

Compositions, reaction mixtures, and methods for performing an amplification reaction, including multiplex amplification reaction, wherein the method comprises using one or more amplification oligomer complexes comprising linked first and second amplification oligomer members. In one aspect, the amplification oligomer complex is hybridized to a target nucleic acid, the target nucleic acid with hybridized amplification oligomer complex is then captured, and other components are washed away. Target sequences of the target nucleic acids are pre-amplified to generate a first amplification product. The first amplification product is amplified in one or more secondary amplification reactions to generate second amplification products.

CROSSREFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/956,158,filed Jul. 31, 2013, allowed, which is a divisional of U.S. applicationSer. No. 12/828,676, filed Jul. 1, 2010, now U.S. Pat. No. 8,512,955,which claims the benefit of priority of U.S. Provisional Application No.61/222,150, filed on Jul. 1, 2009, the contents of each are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to molecular biology, more specifically to invitro amplification of nucleic acids, which is useful for increasing thenumber of copies of a nucleic acid sequence to provide sufficient copiesto be readily detected.

BACKGROUND

Nucleic acid amplification provides a means for making more copies of anucleic acid sequence that is relatively rare or unknown, foridentifying the source of nucleic acids, or for making sufficientnucleic acid to provide a readily detectable amount. Amplification isuseful in many applications, for example, in diagnostics, drugdevelopment, forensic investigations, environmental analysis, and foodtesting. Many methods for amplifying nucleic acid sequences in vitro areknown, including polymerase chain reaction (PCR), ligase chain reaction(LCR), replicase-mediated amplification, strand-displacementamplification (SDA), “rolling circle” types of amplification, andvarious transcription associated amplification methods. These knownmethods use different techniques to make amplified sequences, whichusually are detected by using a variety of methods. PCR amplificationuses a DNA polymerase, oligonucleotide primers, and thermal cycling tosynthesize multiple copies of both strands of a double-stranded DNA(dsDNA) or dsDNA made from a cDNA (U.S. Pat. Nos. 4,683,195, 4,683,202,and 4,800,159). LCR amplification uses an excess of two complementarypairs of single-stranded probes that hybridize to contiguous targetsequences and are ligated to form fused probes complementary to theoriginal target, which allows the fused probes to serve as a templatefor further fusions in multiple cycles of hybridization, ligation, anddenaturation (U.S. Pat. No. 5,516,663 and EP 0320308 B1).Replicase-mediated amplification uses a self-replicating RNA sequenceattached to the analyte sequence and a replicase, such asQ.beta.-replicase, to synthesize copies of the self-replicating sequencespecific for the chosen replicase, such as a Q.beta. viral sequence(U.S. Pat. No. 4,786,600). The amplified sequence is detected as asubstitute or reporter molecule for the analyte sequence. SDA uses aprimer that contains a recognition site for a restriction endonucleasewhich allows the endonuclease to nick one strand of a hemimodified dsDNAthat includes the target sequence, followed by a series of primerextension and strand displacement steps (U.S. Pat. Nos. 5,422,252 and5,547,861). Rolling circle types of amplification rely on a circular orconcatenated nucleic acid structure that serves as a template used toenzymatically replicate multiple single-stranded copies from thetemplate (e.g., U.S. Pat. Nos. 5,714,320 and 5,834,252). Transcriptionassociated amplification refers to methods that amplify a sequence byproducing multiple transcripts from a nucleic acid template. Suchmethods generally use one or more oligonucleotides, of which oneprovides a promoter sequence, and enzymes with RNA polymerase and DNApolymerase activities to make a functional promoter sequence near thetarget sequence and then transcribe the target sequence from thepromoter (e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, 5,130,238,5,437,990, 4,868,105 and 5,124,246, PCT Pub. WO 1988/010315 A1 and USPub. 2006-0046265 A1). Nucleic acid amplification methods may amplify aspecific target sequence (e.g., a gene sequence), a group of relatedtarget sequences, or a surrogate sequence, which may be referred to as atag or reporter sequence that is amplified and detected in place of theanalyte sequence. The surrogate sequence is only amplified if theanalyte target sequence is present at some point during the reaction.Modified nucleic acid amplification methods may amplify more than onepotential target sequence by using “universal” primer(s) or universalpriming. One form of PCR amplification uses universal primers that bindto conserved sequences to amplify related sequences in a PCR reaction(Okamoto et al., 1992, J. Gen. Virol. 73 (Pt. 3):673-9, Persing et al,1992, J. Clin. Microbiol. 30(8):2097-103). Methods that use universalprimers often are paired with use of a species-specific, gene-specificor type-specific primer or primers to generate an amplified sequencethat is unique to a species, genetic variant, or viral type, which maybe identified by sequencing or detecting some other characteristic ofthe amplified nucleic acid. Anchored PCR is another modified PCR methodthat uses a universal primer or an “adapter” primer to amplify asequence that is only partially known. Anchored PCR introduces an“adaptor” or “universal” sequence into a cDNA and then uses a primerthat binds to the introduced sequence in subsequent amplification steps.Generally, anchored-PCR uses a primer directed to a known sequence tomake a cDNA, adds a known sequence (e.g., poly-G) to the cDNA or uses acommon sequence in the cDNA (e.g., poly-T), and performs PCR by using auniversal primer that binds to the added or common sequence in the cDNAand a downstream target-specific primer (Loh et al., 1989, Science243(4888):217-20; Lin et al., 1990, Mol. Cell. Biol. 10(4):1818-21).Nested PCR may use primer(s) that contain a universal sequence unrelatedto the analyte target sequence to amplify nucleic acid from unknowntarget sequences in a reaction (Sullivan et al, 1991, Electrophoresis12(1):17-21; Sugimoto et al., 1991, Agric. Biol. Chem. 55(11):2687-92).

Chamberlain, et al., (Nucleic Acid Research, (1988) 16:11141 11156)first demonstrated multiplex PCR analysis for the human dystrophin gene.Multiplex reactions are accomplished by careful selection andoptimization of specific primers. Developing robust, sensitive andspecific multiplex reactions have demanded a number of specific designconsiderations and empiric optimizations. This results in longdevelopment times and compromises reaction conditions that reduce assaysensitivity. In turn, development of new multiplex diagnostic testsbecomes very costly. A number of specific problems have been identifiedthat limit multiplex reactions. Incorporating primer sets for more thanone target requires careful matching of the reaction efficiencies. Ifone primer amplifies its target with even slightly better efficiency,amplification becomes biased toward the more efficiently amplifiedtarget resulting in inefficient amplification, varied sensitivity andpossible total failure of other target genes in the multiplex reaction.This is called “preferential amplification.” Preferential amplificationcan sometimes be corrected by carefully matching all primer sequences tosimilar lengths and GC content and optimizing the primer concentrations,for example by increasing the primer concentration of the less efficienttargets. Incorporation of inosine into primers in an attempt to adjustthe primer amplification efficiencies (Wu, et al., U.S. Pat. No.5,738,995 (1998)) has also been used. Another approach is to designchimeric primers, wherein each primer contains a 3′ region complementaryto sequence-specific target recognition and a 5′ region made up of auniversal sequence. Using the universal sequence primer permits theamplification efficiencies of the different targets to be normalized.See, Shuber, et al., Genome Research, (1995) 5:488 493; and U.S. Pat.No. 5,882,856. Chimeric primers have also been utilized to multiplexisothermal strand displacement amplification (U.S. Pat. Nos. 5,422,252,5,624,825, and 5,736,365). Since multiple primer sets are present inmultiplex amplification reactions, multiplexing is frequentlycomplicated by artifacts resulting from cross-reactivity of the primers.All possible combinations must be analyzed so that as the number oftargets increases this becomes extremely complex and severely limitsprimer selection. Even carefully designed primer combinations oftenproduce spurious products that result in either false negative or falsepositive results. The reaction kinetics and efficiency is altered whenmore than one reaction is occurring simultaneously. Each multiplexedreaction for each different specimen type must be optimized forMgCl.sub.2 concentration and ratio to the deoxynucleotide concentration,KCl concentration, amplification enzyme concentration, and amplificaiotnreaction times and temperatures. There is competition for the reagentsin multiplex reactions so that all of the reactions plateau earlier. Asa consequence, multiplexed reactions in general are less sensitive thanthe corresponding uniplex reaction. Another consideration tosimultaneous amplification reactions is that there must be a method forthe discrimination and detection of each of the targets. The number ofmultiplexed targets is then further limited by the number of dye orother label moieties distinguishable within the reaction. As the numberof different fluorescent moieties to be detected increases, so does thecomplexity of the optical system and data analysis programs necessaryfor result interpretation. An approach is to hybridize the amplifiedmultiplex products to a solid phase then detect each target. This canutilize a planar hybridization platform with a defined pattern ofcapture probes (U.S. Pat. No. 5,955,268), or capture onto a beadset thatcan be sorted by flow cytometry (U.S. Pat. No. 5,981,180). Due to thesummation of all of the technical issues discussed, current technologyfor multiplex gene detection is costly and severely limited in thenumber and combinations of genes that can be analyzed. Generally, thesereactions multiplex only two or three targets with a maximum of aroundten targets. Isothermal amplification reactions are more complex thanPCR and even more difficult to multiplex. See, Van Deursen, et al.,Nucleic Acid Research, (1999) 27:e15. U.S. Pat. No. 6,605,451 disclosesa two-step PCR multiplex reaction wherein a small amount of each primerpair is added into a first PCR reaction mix and a first amplification isperformed to increase the amount of target nucleic acids in thereaction. The first reaction is stopped mid log phase and is thenseparated into second reactions each containing primer pairs for one ofthe target nucleic acids. A full amplification is then performed. Thougha limited amount of each of the multiplex primer pairs is present in thefirst reaction, the above discussed problems common to multiplexing arestill present. Further, these various primer pair species can alltransfer into the secondary amplification reactions, causing commonmultiplex problems there as well. There is still a need, therefore, fora method, which permits multiplexing of large numbers of targets withoutextensive design and optimization constraints, and which avoids problemscommon to multiplexing in the presence of a plurality of differentamplifications oligomer pairs. There is also a further need for a methodof detecting a significantly larger number of gene targets from a smallquantity of initial target nucleic acid.

SUMMARY OF THE INVENTION

The current invention is directed to compositions, methods and kits forperforming amplification reactions, and preferably multiplexamplification reactions. In one embodiment, there is a compositioncomprising a first amplification oligomer member linked to a secondamplification oligomer member to form an amplification oligomer complex.In one aspect, the first and second amplification oligomer members are aprimer amplification oligomer and a promoter primer or promoter provideramplification oligomer. In one aspect, the first and secondamplification oligomer members are a first primer amplification oligomerand a second primer amplification oligomer. In one aspect, the first andsecond amplification oligomer members are a promoter primer or promoterprovider amplification oligomer and a primer amplification oligomer. Inone aspect, the first and second oligomer members forming the complexare linked together directly. In another aspect, the first and secondamplification oligomer members are a primer amplification oligomer and apromoter primer or promoter provider amplification oligomer, and thefirst and second oligomer members forming the complex are linkedtogether indirectly. In one aspect, one or both of the first and secondoligomer members forming the complex comprises a universal tag sequenceregion. A universal tag sequence region is useful for allowingsubsequent amplification to occur using a universal amplificationoligomer targeting that universal tag sequence region.

In one embodiment, there is a method for performing an amplificationreaction wherein the method comprises one or more amplification oligomercomplexes comprising linked first and second amplification oligomermembers. In one aspect of the method, the amplification oligomer complexis hybridized to a target nucleic acid, the target nucleic acid withhybridized amplification oligomer complex is then captured, and othercomponents are washed away. In one aspect, target capture is performedusing a target capture oligomer, an immobilized probe oligomer and asolid support such as a magnetic bead. In this aspect, the capturedtarget nucleic acid and hybridized amplification oligomer complex isseparated from other sample components using a magnetic bead capturesystem, is washed and is then resuspended. In one aspect, the capturedtarget nucleic acid and hybridized amplification oligomer complex isresuspended into an amplification reaction mixture. In another aspect,the captured target nucleic acid and hybridized amplification oligomercomplex is resuspended into a pre-amplification reaction mixture. In anembodiment of the method wherein the target nucleic acid is resuspendedinto a pre-amplification reaction, the pre-amplification reactionmixture is an oligomer-less reaction mixture. In one aspect, thepre-amplification reaction mixture comprises a reverse transcriptaseand/or an RNA polymerase. The pre-amplification reaction step, then,includes forming of a complementary strand of the target nucleic acidand generating RNA transcripts therefrom. In embodiments wherein theamplification oligomer complex comprises first and second amplificationoligomers wherein one or both of these oligomers comprises an universaltag sequence region, then the RNA transcripts generated therefrom willcomprise a universal sequence region or complement thereof. Followingpre-amplification, initial/first amplification product in thepre-amplified sample is then amplified in a secondary amplificationreaction. The secondary amplification reaction mixtures comprise eithertarget specific amplification oligomers, universal amplificationoligomer or both. Secondary amplifications are preferably performed bytransferring all or a part of the pre-amplified product to one or morereaction wells containing an amplification reaction mixture. In thesecondary amplification reaction a secondary amplification product isgenerated from template transferred from the pre-amplified sample.Amplified product is detected by any of a number of detection methods.In one aspect, the detection step is a detection probe based detectionstep. In another aspect, the detection step is a real-time detectionstep. In another aspect, the detection step is a sequencing step. Inanother aspect, the detection step uses mass spectrometry.

In one embodiment, there is a method for performing a multiplexamplification reaction wherein the method comprises two or moreamplification oligomer complexes comprising linked first and secondamplification oligomer members, and wherein each of the two or moreamplification oligomers is configured to hybridize to a different targetnucleic acid. In one aspect of the method, the amplification oligomercomplexes are hybridized to their target nucleic acids, the targetnucleic acids with hybridized amplification oligomer complexes are thencaptured, and other components are washed away, including unhybridizedamplification oligomer complexes. The target capture step is performedusing a target capture oligomer, an immobilized probe oligomer and asolid support such as a magnetic bead. Following magnetic bead captureand wash, the target nucleic acids and hybridized amplification oligomercomplexes are resuspended into a pre-amplification reaction mixture. Thepre-amplification reaction mixture is an oligomer-less reaction mixture,preferably comprising a reverse transcriptase and/or an RNA polymerase.For each of the target nucleic acids in the pre-amplification, thehybridized amplification oligomer complex is used by the enzymes tofirst form complementary strands for each of the target nucleic acids,and to then generate RNA transcripts therefrom. Followingpre-amplification, initial/first amplification product in thepre-amplified sample is then split into two ore more secondaryamplification reactions, each of which comprises target specificamplification oligomers for one of the target nucleic acid RNA products.In the secondary amplification reaction a secondary amplificationproduct is generated from template transferred from the pre-amplifiedsample. Amplification is performed and the amplified product is detectedby any of a number of detection methods. In one aspect, the detectionstep is a detection probe based detection step. In another aspect, thedetection step is a real-time detection step. In another aspect, thedetection step is a sequencing step. In another aspect, the detectionstep uses mass spectrometry.

In one embodiment, an amplification oligomer complex composition isdisclosed that includes a target specific universal (TSU) promoteroligonucleotide that includes a 5′ promoter sequence, an internal firstuniversal sequence (U1), and a 3′ first target specific sequence (TS1)that binds specifically to a target sequence contained in a targetnucleic acid, wherein the TSU promoter oligonucleotide is a TSU promoterprimer that has a 3′ terminus that is capable of being extended by apolymerase, or is a TSU promoter provider oligonucleotide that has ablocked 3′ terminus that is incapable of being extended by a polymerase,a TSU non-promoter primer oligonucleotide made up of a 5′ seconduniversal sequence (U2) and a 3′ second target specific sequence (TS2)which is different from the TS1, and a means for directly or indirectlyjoining the TSU promoter oligonucleotide to the TSU non-promoter primeroligonucleotide, thereby forming a target specific universal (TSU)primer complex or TSU-complex.

In another embodiment, an amplification oligomer complex composition isdisclosed that includes a first promoter oligonucleotide that includes a5′ promoter sequence, and a 3′ first target specific sequence (TS1) thatbinds specifically to a target sequence contained in a target nucleicacid, wherein the promoter oligonucleotide is a promoter primer that hasa 3′ terminus that is capable of being extended by a polymerase, or is apromoter provider oligonucleotide that has a blocked 3′ terminus that isincapable of being extended by a polymerase, a second non-promoterprimer oligonucleotide made up of a second target specific sequence(TS2) which is different from the TS1, and a means for directly orindirectly joining the promoter oligonucleotide to the non-promoterprimer oligonucleotide, thereby forming an amplification oligomercomplex. In one aspect, the means for means for joining the promoteroligonucleotide to the non-promoter primer oligonucleotide is anindirect means. In another aspect, the means for means for joining thepromoter oligonucleotide to the non-promoter primer oligonucleotide is adirect hybridization, wherein the non-promoter primer oligomer comprisesa 3′ target specific sequence and a 5′ nucleic acid sequence that issubstantially complementary to all or a portion of the promoter sequenceof the first promoter oligomer, thereby forming a DH-complex.

In one embodiment, the means for directly joining the promoteroligonucleotide to the non-promoter primer oligonucleotide is a covalentlinkage. In another embodiment, the covalent linkage is formed via apolynucleotide linker sequence, which may be a covalent linkage formedvia a non-nucleotide abasic linker compound. Another embodiment uses ameans for indirectly joining the promoter oligonucleotide to thenon-promoter primer oligonucleotide that is a non-covalent linkage ofmembers of a binding pair to join the promoter oligonucleotide and thenon-promoter primer oligonucleotide to a support, in which one member ofthe binding pair is present on the promoter oligonucleotide or thenon-promoter primer oligonucleotide and the other member of the bindingpair is attached to the support. In another embodiment, the means fordirectly joining the promoter oligonucleotide to the non-promoter primeroligonucleotide is a hybridization complex between a first sequence onthe promoter oligonucleotide and a second sequence on the non-promoterprimer that is complementary to the first sequence on the promoteroligonucleotide. The means for indirectly joining the promoteroligonucleotide to the non-promoter primer oligonucleotide may be ahybridization complex that includes an S-oligonucleotide that contains afirst sequence complementary to a sequence in the promoteroligonucleotide and a second sequence complementary to a sequence in thenon-promoter primer oligonucleotide. In one embodiment theS-oligonucleotide contains a first sequence complementary to theuniversal sequence in the promoter oligonucleotide and theS-oligonucleotide contains a second sequence complementary to theuniversal sequence in the non-promoter primer oligonucleotide. Thecomposition may also include a target specific capture oligonucleotidethat contains a sequence that hybridizes specifically to a sequence inthe target nucleic acid. The target specific capture oligonucleotide mayhybridize to the target nucleic acid at a target sequence that isdifferent from the target sequence hybridized by the promoteroligonucleotide TS sequence or by the non-promoter primer TS sequence.The target capture oligonucleotide may contain a means for binding thetarget nucleic acid to a support. The composition may also include auniversal promoter primer made up a 5′ promoter sequence and a 3′universal sequence that is the same as the universal sequence of thepromoter oligonucleotide. Another embodiment is a composition thatfurther includes a universal primer made up a universal sequence that isthe same as the universal sequence of the non-promoter primeroligonucleotide. The composition may also include a blockeroligonucleotide that hybridizes specifically to a sequence in a targetnucleic acid strand that is completely different, partially different orthe same as the sequence in the target nucleic acid strand that the TSsequence of the promoter oligonucleotide or the TS sequence of thenon-promoter primer oligonucleotide binds, and wherein the blockeroligonucleotide has a 3′ blocked terminus that is not capable of beingextended by a polymerase. In some embodiments that include anS-oligonucleotide, it is made up of (1) a first terminal region sequencethat is complementary to the U1 sequence of the promoter primer and (2)a second terminal region sequence that is complementary to the U2sequence of the TSU non-promoter primer, and (3) a linking moiety thatlinks the first and second terminal region sequences. The linking moietymay be a non-nucleic acid chemical compound that covalently links thefirst and second terminal region sequences. The composition may alsoinclude at least one universal promoter primer made up of a 5′ promotersequence and a 3′ U1 sequence and at least one target specific primer(TSP) made up of a sequence that is complementary to a sequencecontained in an RNA transcript made from a double stranded DNA thatcontains a cDNA sequence made from synthetic extension of the 3′ end ofthe promoter primer oligonucleotide.

Also disclosed is a method of amplifying a target nucleic acidcomprising the steps of: isolating a target nucleic acid from a mixtureby binding to the target nucleic acid a target capture probe that bindsspecifically to the target nucleic acid and provides a means forattaching the bound target nucleic acid to a support that is separatedfrom the mixture, and further hybridizing to the target nucleic acid inthe mixture a amplification oligomer complex made up of (A)(1) a TSUpromoter primer oligonucleotide that includes a 5′ promoter sequence, aninternal first universal sequence (U1), and a 3′ first target specificsequence (TS1) that binds specifically to a target sequence contained ina target nucleic acid, and a 3′ terminus that is capable of beingextended by a polymerase, (2) a TSU non-promoter primer oligonucleotidemade up of a 5′ second universal sequence (U2) and a 3′ second targetspecific sequence (TS2) which is different from the TS1, and (3) a meansfor directly or indirectly joining the TSU promoter oligonucleotide tothe TSU non-promoter primer oligonucleotide; or (B)(1) a promoter primeroligonucleotide that includes a 5′ promoter sequence, and a 3′ firsttarget specific sequence (TS1) that binds specifically to a targetsequence contained in a target nucleic acid, and a 3′ terminus that iscapable of being extended by a polymerase, (2) a non-promoter primeroligonucleotide comprising at least a second target specific sequence(TS2) which is different from the TS1, and (3) a means for directly orindirectly joining the promoter oligonucleotide to the non-promoterprimer oligonucleotide. The method includes hybridizing the promoterprimer oligonucleotide to a target sequence in the target nucleic acidvia a TS sequence in the promoter primer, synthetically extending the 3′terminus of the promoter primer oligonucleotide hybridized to the targetnucleic acid by using a polymerase in vitro nucleic acid synthesis inwhich the target nucleic acid is a template to make a first cDNA strand,hybridizing the non-promoter primer oligonucleotide to the first cDNAstrand by specific hybridization of the TS sequence in the non-promoterprimer oligonucleotide to a target sequence contained in the first cDNAstrand, synthetically extending the 3′ terminus of the non-promoterprimer oligonucleotide hybridized to the first cDNA strand by apolymerase in vitro nucleic acid synthesis to made a second DNA strand,thereby making a substantially double-stranded DNA. Depending onewhether complex A or complex B, described directly above, is used as theamplification oligomer complex, then the double stranded DNA willcontain a functional promoter sequence and, optionally, the U1 sequence.RNA transcripts are then enzymatically transcribed from the functionalpromoter sequence of the substantially double-stranded DNA. The RNAtranscripts comprise a first target specific sequence (TS1), a secondtarget specific sequence (TS2′). Further, when the amplificationoligomer complex at A is used that the RNA transcripts further comprisea 5′ U1 region sequence, and a 3′ universal sequence (U2′) that iscomplementary to the U2 sequence. The method further comprisesamplification either using target specific amplification oligomers or,if the U1 and/or U2′ sequences are present in the RNA transcript, usinga universal primer oligonucleotide (UP2) that contains a universalsequence U2 to the RNA transcript at the U2′ sequence. Amplification ispreformed under isothermal conditions, synthetically extending the 3′terminus of primer oligomer (either universal or target specific,depending on the amplification oligomer complex used) by enzymatic invitro nucleic acid synthesis to make a cDNA strand, and enzymaticallyremoving the RNA transcript strand, hybridizing a promoter primeroligonucleotide (either universal or target specific) to the cDNA madein the previous step under isothermal conditions, syntheticallyextending at the 3′ terminus by enzymatic in vitro nucleic acidsynthesis to make a dsDNA that contains a functional promoter, andtranscribing multiple RNA transcripts from the functional promoter ofthe dsDNA, which are amplification products that may serve as templatesfor further enzymatic in vitro nucleic acid synthesis under isothermalconditions. The method may also include the step of detecting theamplification products to indicate the presence of an analyte in themixture from which the target nucleic acid was isolated. Detection canbe real-time detection and/or quantitative detection.

Another disclosed method of amplifying a target nucleic acid includesisolating a target nucleic acid from a mixture by binding to the targetnucleic acid a target capture probe that binds specifically to thetarget nucleic acid and provides a means for attaching the bound targetnucleic acid to a support that is separated from the mixture, andfurther hybridizing to the target nucleic acid in the mixture anamplification oligomer complex made up of (A)(1) a TSU promoteroligonucleotide that includes a 5′ promoter sequence, an internal firstuniversal sequence (U1), and a 3′ first target specific sequence (TS1)that binds specifically to a target sequence contained in a targetnucleic acid, wherein the TSU promoter oligonucleotide is a TSU promoterprovider oligonucleotide that has a blocked 3′ terminus that isincapable of being extended by a polymerase, (2) a TSU non-promoterprimer oligonucleotide made up of a 5′ second universal sequence (U2)and a 3′ second target specific sequence (TS2) which is different fromthe TS1, and (3) a means for directly or indirectly joining the TSUpromoter oligonucleotide to the TSU non-promoter primer oligonucleotide;or (B)(1) a promoter oligonucleotide that includes a 5′ promotersequence and a 3′ first target specific sequence (TS1) that bindsspecifically to a target sequence contained in a target nucleic acid,wherein the TSU promoter oligonucleotide is a TSU promoter provideroligonucleotide that has a blocked 3′ terminus that is incapable ofbeing extended by a polymerase, (2) a TSU non-promoter primeroligonucleotide comprising at least a 3′ second target specific sequence(TS2) which is different from the TS1, and (3) a means for directly orindirectly joining the promoter oligonucleotide to the non-promoterprimer oligonucleotide. The method steps also include hybridizing thenon-promoter primer oligonucleotide to a target sequence in the targetnucleic acid via the TS sequence in the non-promoter primer, optionallyhybridizing a blocker oligonucleotide with a 3′ blocked end that isincapable of being extended synthetically by a polymerase to a sequenceon the target nucleic acid that is in the 5-primer direction away fromthe position that the non-promoter primer oligonucleotide hybridizes inthe target nucleic acid, synthetically extending the 3′ terminus of thenon-promoter primer hybridized to the target nucleic acid by using apolymerase in vitro nucleic acid synthesis in which the target nucleicacid is a template to make a first cDNA strand, hybridizing the promoterprovider oligonucleotide to the first cDNA strand by specifichybridization of the TS sequence in the promoter provideroligonucleotide to a target sequence contained in the first cDNA strand,synthetically extending the 3′ terminus of the first cDNA by usingsequence in the promoter provider as a template to make a substantiallydouble-stranded DNA that contains a functional promoter sequence and,when the amplification oligomer complex at A is used, described directlyabove, further comprises the U1 sequence. RNA transcripts are thenenzymatically transcribed from the functional promoter sequence of thesubstantially double-stranded DNA. The RNA transcripts comprise a firsttarget specific sequence (TS1), a second target specific sequence(TS2′). Further, when the amplification oligomer complex at A is usedthat the RNA transcripts further comprise a 5′ U1 region sequence, and a3′ universal sequence (UT) that is complementary to the U2 sequence. Themethod further comprises amplification either using target specificamplification oligomers or, if the U1 and/or U2′ sequences are presentin the RNA transcript, using a universal primer oligonucleotide (UP2)that contains a universal sequence U2 to the RNA transcript at the UTsequence. Amplification is preformed under isothermal conditions,synthetically extending the 3′ terminus of the primer oligomer, eitheruniversal or target specific, by enzymatic in vitro nucleic acidsynthesis to made a cDNA strand, and enzymatically removing the RNAtranscript strand, hybridizing a promoter oligonucleotide, eitheruniversal or target specific, that contains a promoter sequence,optionally, a universal sequence U1, and a target specific sequence witha 3′ blocked end to the cDNA made in the previous step and, underisothermal conditions, synthetically extending the 3′ terminus of thecDNA to make a functional double-stranded promoter and transcribingmultiple RNA transcripts from the functional promoter of the dsDNA.These RNA transcripts are amplification products that may serve astemplates for further enzymatic in vitro nucleic acid synthesis underisothermal conditions. The method may further include the step ofdetecting the amplification products to indicate the presence of ananalyte in the sample from which the target nucleic acid was isolated.

Also discloses is a method of amplifying a target nucleic acid thatincludes steps of isolating a target nucleic acid from a mixture bybinding to the target nucleic acid a target capture probe that bindsspecifically to the target nucleic acid and provides a means forattaching the bound target nucleic acid to a support that is separatedfrom the mixture and further hybridizing to the target nucleic acid inthe mixture a target specific universal (TSU) promoter primeroligonucleotide that includes a 5′ promoter sequence, an internal firstuniversal sequence (U1), and a 3′ first target specific sequence (TS1)that binds specifically to a target sequence contained in a targetnucleic acid, and a 3′ terminus that is capable of being extended by apolymerase, synthetically extending the 3′ terminus of the TSU promoterprimer oligonucleotide hybridized to the target nucleic acid by using apolymerase in vitro nucleic acid synthesis in which the target nucleicacid is a template to make a first cDNA strand, adding to theamplification reaction mixture a target specific (TS) non-promoterprimer that contains a second target specific sequence (TS2) which isdifferent from the TS1, hybridizing the TS non-promoter primeroligonucleotide to the first cDNA strand by specific hybridization ofthe TS2 sequence to a target sequence contained in the first cDNAstrand, synthetically extending the 3′ terminus of the TS non-promoterprimer oligonucleotide hybridized to the first cDNA strand by apolymerase in vitro nucleic acid synthesis to made a second DNA strand,thereby making a substantially double-stranded DNA that contains afunctional promoter sequence and the U1 sequence, enzymaticallytranscribing RNA transcripts from the functional promoter sequence ofthe substantially double-stranded DNA to make RNA transcripts thatcontain a 5′ U1 region sequence, a first target specific sequence (TS1),a second target specific sequence (TS2′), hybridizing a universalpromoter primer oligonucleotide that contains a universal sequence U1′to the RNA transcript at the U1 sequence, under isothermal conditions,synthetically extending the 3′ terminus of the universal promoter primerby enzymatic in vitro nucleic acid synthesis to made a cDNA strand, andenzymatically removing the RNA transcript strand, hybridizing a TSnon-promoter primer oligonucleotide to a specific sequence in the cDNAmade in the previous step, under isothermal conditions, syntheticallyextending the 3′ terminus of the TS non-promoter primer by enzymatic invitro nucleic acid synthesis to made a dsDNA that contains a functionalpromoter, and transcribing multiple RNA transcripts from the functionalpromoter of the dsDNA, which transcripts are amplification products thatmay serve as templates for further enzymatic in vitro nucleic acidsynthesis under isothermal conditions by repeating the synthetic steps.The method may further include detecting the amplification products toindicate the presence of an analyte in the mixture from which the targetnucleic acid was isolated.

Another disclosed method of amplifying a target nucleic acid includesthe steps of isolating a target nucleic acid from a mixture by bindingto the target nucleic acid a target capture probe that bindsspecifically to the target nucleic acid and provides a means forattaching the bound target nucleic acid to a support that is separatedfrom the mixture and further hybridizing to the target nucleic acid inthe mixture a TSU non-promoter primer oligonucleotide made up of a 5′universal sequence (U2) and a 3′ target specific sequence (TS2),hybridizing the TSU non-promoter primer oligonucleotide to a targetsequence in the target nucleic acid via the TS2 sequence to acomplementary sequence in the target nucleic acid, hybridizing a blockeroligonucleotide with a 3′ blocked end that is incapable of beingextended synthetically by a polymerase to a sequence on the targetnucleic acid that is downstream from the position that the TSUnon-promoter primer oligonucleotide hybridizes in the target nucleicacid, synthetically extending the 3′ terminus of the TSU non-promoterprimer hybridized to the target nucleic acid by using a polymerase invitro nucleic acid synthesis in which the target nucleic acid is atemplate to make a first cDNA strand, hybridizing to the first cDNAstrand a target specific TS promoter provider oligonucleotide thatincludes a 5′ promoter sequence and a 3′ target specific sequence (TS1)that binds specifically to a target sequence contained in a targetnucleic acid, and a blocked 3′ terminus that is incapable of beingextended by a polymerase, by specific hybridization of the TS1 sequenceto a complementary sequence in the first cDNA strand, syntheticallyextending the 3′ terminus of the first cDNA by using sequence in the TSpromoter provider as a template to make a substantially double-strandedDNA that contains a functional promoter sequence and a TS1 sequence,enzymatically transcribing RNA transcripts from the functional promotersequence to make RNA transcripts that contain a 5′ target specificsequence TS1, a target specific sequence TS2′ and a U2′ sequence,hybridizing a universal primer oligonucleotide (UP2) that contains auniversal sequence U2 to the RNA transcript at the UT sequence, underisothermal conditions, synthetically extending the 3′ terminus of theUP2 by enzymatic in vitro nucleic acid synthesis to made a cDNA strand,and enzymatically removing the RNA transcript strand, hybridizing a TSpromoter provider oligonucleotide that contains a promoter sequence anda 3′ blocked end to the cDNA made in the previous step, under isothermalconditions, synthetically extending the 3′ terminus of the cDNA to makea functional double-stranded promoter by using the TS promoter provideroligonucleotide as a template and by enzymatic in vitro nucleic acidsynthesis to made a dsDNA that contains a functional promoter, andtranscribing multiple RNA transcripts from the functional promoter ofthe dsDNA, which transcripts are amplification products that may serveas templates for further enzymatic in vitro nucleic acid synthesis underisothermal conditions by repeating the synthetic steps. The method mayalso include detecting the amplification products to indicate thepresence of an analyte in the sample from which the target nucleic acidwas isolated.

One embodiment is a method for simultaneously amplifying at least twodifferent target nucleic acid sequences contained in a sample comprisingthe steps of: contacting a sample with at least two amplificationoligomer complexes that hybridize to different target nucleic acidsequences, wherein each of the amplification oligomer complexescomprises a first amplification oligomer member that is directly joinedto a second amplification oligomer member; pre-amplifying the targetnucleic acid sequences using the amplification oligomer complexes,thereby generating first amplification products for each target nucleicacid hybridized by an amplification oligomer complex; splitting thepre-amplified sample into at least two separate secondary targetspecific amplification reactions; amplifying the pre-amplified samplegenerated in the pre-amplification reaction above using target specificamplification oligomers, thereby generating second amplificationproducts. In this embodiment, the amplification oligomer complexes areconfigured to hybridize with different target nucleic acid sequences. Inthe pre-amplification step, an amount of first amplification product isgenerated, thereby increasing the amount of each target nucleic acidsequence that is available for secondary amplification in separatecontainers. The secondary amplification containers perform a specificamplification, either by way of target specific amplification primers inthat secondary amplification reaction, and/or by target specificdetection probes. Separate target specific amplification reactions avoidthe problems encountered with multiplex amplification, e.g., primerdimers, different amplification efficiencies, target biased reagentconsumption, mis-priming, cross-reactivity and other known problems.

In one aspect of this embodiment the sample is contacted with at leasttwo amplification oligomer complexes and at least two target captureoligomers that hybridize to different target nucleic acid sequences. Inone aspect, the at least two amplification oligomer complexes and the atleast two target capture oligomers are contacted to the sample underconditions for hybridizing the at least two amplification oligomercomplexes and the at least two target capture oligomers to theirrespective different target nucleic acid sequences present in thesample. In one aspect, the at least two target capture oligomers arecontacted by a solid support and immobilized probe for performing atarget capture step. In one aspect, a wash step is performed.

In one aspect of this embodiment, target nucleic acid sequenceshybridized to amplification oligomer complexes are contacted with anoligomerless pre-amplification reaction reagent. Thus, thepre-amplification reaction occurs using just the amplification oligomermembers forming the amplification oligomer complex. In one aspect, theoligomerless pre-amplification reaction reagent comprises a reversetranscriptase, an RNA polymerase or both. In one aspect, thepre-amplifying reaction comprises contacting the at least two targetnucleic acid sequences with an oligomerless pre-amplification reactionreagent comprises a reverse transcriptase, an RNA polymerase or both. Inone aspect, the pre-amplification reaction generates from one of the atleast two target nucleic acid sequences, a first amplification productthat is a plurality of RNA transcripts.

In one aspect of this embodiment, the amplifying reaction that generatessecondary amplification products is a quantitative amplificationreaction. In one aspect, the amplifying reaction that generatessecondary amplification products is an exponential amplificationreaction.

In one aspect of this embodiment, a portion of the pre-amplificationreaction is transferred into a secondary target specific amplificationreaction comprising at least one target specific amplification oligomer.In one aspect, an additional portion of the pre-amplification reactionis transferred into a secondary target specific amplification reactioncomprising at least one different target specific amplificationoligomer. The different first amplification products generated from eachdifferent target nucleic acid sequence hybridized to one of thedifferent amplification oligomer complexes is then amplified in aseparate target specific secondary amplification. In another aspect, thesecondary amplification can be a multiplex amplification reaction thatis configured to amplify fewer targets that what is suspected to be inthe sample. For example, if it is suspected that the sample has fourtargets, the pre-amplification reaction will generate up to fourdifferent first amplification products, and then the pre-amplificationreaction can be split into at least one, more preferably two, morepreferably three, more preferably four or more separate secondaryamplification reactions. If just one, then the pre-amplificationreaction was to increase the amount of template for the secondaryamplification reaction and/or to incorporate universal tag sequences. Iftwo or three, then at least one of the secondary amplifications is amultiplex reaction but with fewer than four target nucleotide sequences.Multiplex reactions having fewer targets or optimized pairs can avoidsome of the multiplex problems, this is more true is universal tagsequences have been incorporated. If four, then there can be a separatesecondary amplification reaction for each target suspected in thesample. If more than four, then the first amplification products can bespecifically amplified more than one target sequence and/or can beamplified in duplicate. In one aspect, the first amplification productsare divided into a number of secondary target specific amplificationreactions that is equal to the number of different target nucleic acidsequences suspected of being present in the sample.

In one aspect, the pre-amplifying reaction is accomplished using asubstantially isothermal amplification reaction. In one aspect, thesecondary amplification reaction is accomplished using a substantiallyisothermal amplification reaction. In one aspect, the firstamplification product generated in the pre-amplifying step for a targetnucleic acid hybridized by an amplification oligomer complex is from 100to 10000 RNA transcripts.

In one aspect, the amplification oligomer complex is a firstamplification oligomer indirectly joined to a second amplificationoligomer complex. In one aspect, the amplification oligomer complex is afirst amplification oligomer directly joined to a second amplificationoligomer complex. In an aspect, the first and second amplificationoligomers are joined using a DH-complex. In an aspect, the first andsecond amplification oligomers are joined using an S-oligo. In anaspect, the first and second amplification oligomers are joined using anon-nucleotide linker. In one aspect, the first and second oligomermembers of the amplification oligomer complex are non-promoter primers,promoter primers (with or without blocked 3′-ends), and combinationsthereof. In one aspect, the amplification oligomer complex comprises afirst amplification oligomer member that is a non-promoter primercomprising a target specific sequence joined on its 5′ end to a linkingmember for linking the first amplification oligomer member to a secondamplification oligomer member of the amplification oligomer complex. Inone aspect, the second amplification oligomer member is a promoterprimer, optionally comprising a blocked 3′ terminus. In an aspect, thefirst oligomer member comprises a linking member that is a nucleotidesequence that is complementary to a portion of the nucleotide sequenceof the second amplification oligomer member. In an aspect, the firstoligomer member comprises a linking member that is a nucleotide sequencethat is complementary to the promoter sequence of the secondamplification oligomer member. In one aspect, the amplification oligomercomplex is formed before being added into the pre-amplification reactionmixture. In one aspect, the amplification oligomer complex is formed andthen is added into a target capture reaction mixture. In one aspect, theamplification oligomer complex is formed under hybridizing conditionsduring a target capture step.

In one aspect, a target capture reagent is contacted to the sample,hybridization conditions are provided and the captured target nucleicacids and amplification oligomer complexes are removed from teh samplecomponents and then resuspended in an oligomerless pre-amplificationreagent for pre-amplification.

In one aspect, the pre-amplification reaction method further comprisesusing a blocker oligomer. Blockers are useful in defining the 3′-end ofan amplification product

In one aspect, the secondary amplification product is detected. In oneaspect the first amplification product is detected. Detection can beprobe based, sequencing based, mass-spectrometry based, gelelectrophoresis based, or other common detection technique known in theart. In one aspect, amplifications product from the secondaryamplification reaction is detected using a probe based detection step.In one aspect, the probe based detection step is a real-time detectionstep.

One embodiment is a method for simultaneously amplifying at least twodifferent target nucleic acid sequences contained in a sample comprisingthe steps of: contacting a sample with at least two differentamplification oligomer complexes, wherein each of the amplificationoligomer complexes comprise a first amplification oligomer member with a3′ target specific sequence, joined to a second amplification oligomermember with a 3′ target specific sequence; generating firstamplification products for each target nucleic acid hybridized by anamplification oligomer complex; splitting the first amplificationproducts into at least two separate secondary target specificamplification reactions; and then generating second amplificationproducts. In one aspect, the first amplification oligomer members foreach of the different amplification oligomer complexes, are non-promoterprimers. In one aspect, the first amplification oligomers comprise auniversal nucleotide sequence joined at the 5′ end of the targetspecific sequence (universal tag). In one aspect, each firstamplification oligomer from each different amplification oligomercomplex has a universal nucleotide sequence with substantially similarnucleotide sequences. In one aspect, the first amplification oligomermembers for each of the different amplification oligomer complexes, arepromoter oligonucleotides, optionally blocked at their 3′ ends. In oneaspect, the first amplification oligomers comprise a universalnucleotide sequence joined at the 5′ end of the target specific sequenceand at the 3′ end of the promoter sequence. In one aspect, the firstamplification oligomer from each different amplification oligomercomplex has a universal nucleotide sequence with substantially similarnucleotide sequences. In one aspect, the second amplification oligomermembers for each of the different amplification oligomer complexes, arenon-promoter primers. In one aspect, the second amplification oligomerscomprise a universal nucleotide sequence joined at the 5′ end of thetarget specific sequence. In one aspect, the second amplificationoligomer members for each of the amplification oligomer complexes, arepromoter oligonucleotides, optionally blocked at their 3′ ends. In oneaspect, each second amplification oligomer from each differentamplification oligomer complex has a universal nucleotide sequence withsubstantially similar nucleotide sequences. In one aspect, the differentamplification oligomer complexes are made of first and secondamplification oligomer combinations selected from the group consistingof: two non-promoter primers; two non-promoter primers, one of which hasa universal tag; two non-promoter primers, both of which have auniversal tag; a non-promoter primer and a promoter primer (with orwithout a 3′blocked end); a non-promoter primer and a promoter primer(with or without a 3′blocked end), one of which has a universal tagsequence; a non-promoter primer and a promoter primer (with or without a3′blocked end), both of which have a universal tag sequence; a promoterprimer (with or without a 3′blocked end) and a non-promoter primer; apromoter primer (with or without a 3′blocked end) and a non-promoterprimer, one of which has a universal tag sequence; a promoter primer(with or without a 3′blocked end) and a non-promoter primer, both ofwhich have a universal tag sequence.

In one aspect, the amplification oligomer complex is a firstamplification oligomer indirectly joined to a second amplificationoligomer complex. In one aspect, the amplification oligomer complex is afirst amplification oligomer directly joined to a second amplificationoligomer complex. In an aspect, the first and second amplificationoligomers are joined using a DH-complex. In an aspect, the first andsecond amplification oligomers are joined using an S-oligo. In anaspect, the first and second amplification oligomers are joined using anon-nucleotide linker. In one aspect, the first amplification oligomermember and the second amplification oligomer member of the amplificationoligomer complex are both non-promoter primers joined at their 5′ endsusing a non-nucleotide linker, and wherein at least one of the firstamplification oligomer or the second amplification oligomer has auniversal nucleotide sequence joined at the 5′ end of the targetspecific sequence. In one aspect, the first and second oligomer membersof the amplification oligomer complex are non-promoter primers, promoterprimers (with or without blocked 3′-ends), and combinations thereof. Inone aspect, the amplification oligomer complex comprises a firstamplification oligomer member that is a non-promoter primer comprising atarget specific sequence joined on its 5′ end to a linking member forlinking the first amplification oligomer member to a secondamplification oligomer member of the amplification oligomer complex. Inone aspect, the second amplification oligomer member is a promoterprimer, optionally comprising a blocked 3′ terminus. In an aspect, thefirst oligomer member comprises a linking member that is a nucleotidesequence that is complementary to a portion of the nucleotide sequenceof the second amplification oligomer member. In an aspect, the firstoligomer member comprises a linking member that is a nucleotide sequencethat is complementary to the promoter sequence of the secondamplification oligomer member. In one aspect, the amplification oligomercomplex is formed before being added into the pre-amplification reactionmixture. In one aspect, the amplification oligomer complex is formed andthen is added into a target capture reaction mixture. In one aspect, theamplification oligomer complex is formed under hybridizing conditionsduring a target capture step. In one aspect, the first amplificationoligomer complex is made of a non-promoter primer and a promoter primer,optionally blocked at its 3′ end, wherein the non-promoter primercomprises a nucleotide sequence that is joined to the 5′ end of thetarget specific sequence, and wherein the nucleotide sequence issubstantially the complement of the promoter primer promoter sequenceoligomer member, thereby joining the first and second amplificationoligomer members by hybridizing the complementary promoter sequence tothe promoter sequence.

In one aspect, the first amplification product is generated in asubstantially isothermal amplification reaction. In one aspect, the efirst amplification products are RNA transcripts.

In one aspect, the method includes contacting the sample with at leastone target capture oligomer. In one aspect. at least one target captureoligomer and at least two different amplification oligomer complexeshybridize to a target nucleic acid sequence in the sample, therebycapturing the target nucleic acid sequence, and wherein the targetcapture oligomer:target nucleic acid sequence:amplification oligomercomplex is isolated from other components of the sample. For simplicityin describing these entities, the use of “:” means that the componentsare hybridized together. In one aspect. a wash step is performed.

In one aspect, a target capture oligomer:target nucleic acidsequence:amplification oligomer complex is resuspended in anoligomerless pre-amplification reaction mixture. In one aspect, theoligomerless pre-amplification reaction mixture comprises a polymerase.In one aspect, the oligomerless pre-amplification reaction mixturecomprises a reverse transcriptase. In one aspect, the pre-amplificationreaction mixture comprises an enzyme selected from the group consistingof: a polymerase, an RNA-dependent DNA polymerase, a DNA-dependent DNApolymerase, a DNA-dependent DNA polymerase, a reverse transcriptase, anRNase, or a combination thereof.

In one aspect, the amplification oligomer complex comprises twoamplification oligomer members selected from the group consisting of:non-promoter primers and promoter primers, optionally blocked at the 3′end, wherein at least one of the amplification oligomer memberscomprises a universal tag sequence, the first amplification products aresplit into at least two separate secondary target specific amplificationreactions, wherein the secondary target specific amplification reactionincludes at least one primer targeting all or a portion of the universaltag sequence. In one aspect, the secondary amplification products aredetected using a target specific detection probe oligomer. In oneaspect, the secondary amplification products are detected in real-time.

In one aspect, at least two different amplification oligomer complexesare configured to hybridize two different target nucleic acid sequenceson the same target nucleic acid. In this aspect, it can be determinedwhether two amplification products are part of the same target nucleicacid or are part of separate target nucleic acids. For example, whetheran insertion element has integrated into a genome, as is the case withantibiotic resistance genes integrating into bacterial genomes. Also,fusions can be determined, such as promoter sequence fused to anoncogene. By capturing, the single target nucleic acid that can compriseone or two of these target sequences, hybridizing the differentamplification oligomer complexes, removing the hybridized complex fromother sample components, and then performing an amplification reaction,it can be determined whether both target nucleotide sequence are on asingle target nucleic acid by detecting the presence of one or bothamplification products. Both products means both target nucleotidesequence were present on the same target nucleic acid. In one aspect, atarget capture oligomer is hybridized to the target nucleic acid and thetarget capture oligomer:target nucleic acid:two different amplificationoligomer complexes is separated from other components in the sample. Inone aspect, a first target nucleic acid sequence is an insertionsequence in the target nucleic acid and a second target nucleic acidsequence is not an insertion sequence in the target nucleic acid, andwherein the pre-amplification reaction generates first amplificationproducts from the non-insertion sequence and from the insertion sequenceif present in the target nucleic acid. In one aspect, the presence ofthe target nucleic acid sequences can be determined by detecting tehfirst amplification product, or by performing a single secondary targetspecific amplification reaction or by performing two different secondarytarget specific amplification reactions.

In one aspect, a blocker oligomer is used to define the 3′ end of thefirst amplification product.

In one aspect, a first amplification product is detected in a detectingstep. Detection can be probe based, sequencing based, mass-spectrometrybased, gel electrophoresis based, or other common detection techniqueknown in the art. In one aspect, amplifications product from thesecondary amplification reaction is detected using a probe baseddetection step.

In one aspect, a second amplification product is detected in a detectingstep. Detection can be probe based, sequencing based, mass-spectrometrybased, gel electrophoresis based, or other common detection techniqueknown in the art. In one aspect, amplifications product from thesecondary amplification reaction is detected using a probe baseddetection step. In one aspect, the probe based detection step is areal-time detection step.

In one embodiment, there is a target capture reaction mixture for use ina pre-amplification reaction method that is preferably followed by asecondary target specific amplification reaction method, wherein thereaction mixture comprises at least one target nucleic acid and at leastone amplification oligomer complex. In one aspect, the target capturereaction mixture further comprising at least one solid support. In oneaspect, the solid support is a magbead (magnetic bead). In one aspect,the at least one target capture oligomer is a wobble probe. (See, e.g.,U.S. App. Pub. No.: 2008/0286775 A1 describing wobble probes) In oneaspect, the target capture reaction mixture further comprises animidazolium compound in an amount sufficient to provide 0.05M to 4.2Mwhen combined with the sample.

In one embodiment, there is a pre-amplification reaction mixture forgenerating first amplification products, wherein the pre-amplificationreaction mixture comprises an enzyme selected from the group consistingof: a polymerase, an RNA-dependent DNA polymerase, a DNA-dependent DNApolymerase, a DNA-dependent DNA polymerase, a reverse transcriptase, anRNase, or a combination thereof. In one aspect, the reaction mixture isan oligomerless reaction mixture.

In one embodiment, there is a secondary amplification reaction mixturefor generating second amplification products from first amplificationproducts, wherein the secondary amplification reaction mixture comprisesa pair of amplification oligomer selected from the group consisting of:two non-promoter primers; a promoter primer, optionally blocked at its3′-end, and a non-promoter primer, a universal non-promoter primer thathybridizes to a universal tag sequence in a first amplification productand a prompter primer, optionally blocked at it 3′-end; a non-promoterprimer and a promoter primer, optionally blocked at its 3′-end, and thathybridizes to a universal tag sequence in a first amplification product;two non-promoter primers, each that hybridize to universal tag sequencesin a first amplification product; and a universal non-promoter primerthat hybridizes to a first universal tag sequence in a firstamplification product and a promoter primer, optionally blocked at its3′-end, and that hybridizes to a second universal tag sequence in afirst amplification product. The amplification enzymes may be providedas part of the first amplification product being transferred into thesecondary target specific amplification reaction or the amplificationenzymes may be part of the secondary amplification reaction mixture. Inone aspect, the secondary amplification reaction mixture furthercomprises an enzyme selected from the group consisting of: a polymerase,an RNA-dependent DNA polymerase, a DNA-dependent DNA polymerase, aDNA-dependent DNA polymerase, a reverse transcriptase, an RNase, or acombination thereof.

The accompanying drawings, which constitute a part of the specification,illustrate some embodiments of the invention. These drawings, togetherwith the description, serve to explain and illustrate the principles ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The amplification oligomer complexes illustrated in the figures compriseamplification oligomer members containing universal tag sequences, andthus are referred to as TSU-complexes. This is for illustration and notlimitation. Amplification oligomer complexes comprising oligomer membersthat do not contain universal tag sequences, e.g., DH-complexes, are,for the most part similarly configured and used.

FIG. 1 is a schematic drawing showing: a three-component target-specificuniversal (TSU) primer complex that includes a TSU promoter primer madeup of a 5′ promoter sequence (solid line) labeled P, a universalsequence (dashed line) labeled U1, and a 3′ target-specific sequence(double lines) labeled TS1, which is hybridized to an S-oligonucleotide(S-shaped dotted line) that includes a 5′ universal sequence labeled U1′and a 3′ universal sequence labeled U2′, which is hybridized to a TSUnon-promoter primer made up of a 5′ universal sequence (dashed line)labeled U2 and a 3′ target-specific sequence (double line) labeled TS2;a target-specific capture oligonucleotide made up of a 5′target-specific sequence (double line) labeled TS3 and a 3′ binding pairmember (triple line) labeled BPM; a universal promoter primer (UP1) madeup of a 5′ promoter sequence (solid line) labeled P and a 3′ universalsequence (dashed line) labeled U1; and a universal non-promoter primer(UP2) made up of a universal sequence (dashed line) labeled U2.

FIG. 2 is a schematic drawing illustrating target capture in which: (1)target capture reagent (TCR) contains multiple three-componenttarget-specific universal (TSU) primer complexes (see FIG. 1) specificfor three different targets (labeled TSUa, TSUb, TSUc) and captureprobes specific for the three different targets in which the BPM isshown as poly-A sequences (AAA) and the target-specific sequences arelabeled TSa, TSb, and TSc; (2) TCR is mixed with a sample that contains“Target a”, which allows the TSUa primer complex to hybridize to Targeta and the TSa capture probe to hybridize to Target a; (3) the poly-Asequence of the TSa capture probe hybridizes to an immobilized probe(poly-T sequence shown as TTTT) which is attached to a support (shadedcircle), which allows the complex attached to the support to beseparated from the mixture to retrieve the captured target and TSUprimer complex; and (4) the portion containing the unbound TSU primercomplexes (labeled TSUb and TSUb) is discarded as waste.

FIG. 3 is a schematic drawing that illustrates a three-component TSUprimer complex which is attached to a target strand via hybridization ofthe TS1 sequence of the TSU promoter primer to a complementary TS1′sequence in the target nucleic acid, which is attached to a support(shaded circle) via hybridization of the target specific TS3 sequence ofa capture probe to a complementary TS3′ sequence of the target nucleicacid and the poly-A portion of the capture probe is hybridized to animmobilized poly-T probe that is attached to the support. Verticalconnecting lines (∥∥|) indicate sequence hybridization. The TSU primercomplex is made up of the TSU non-promoter primer hybridized at its U2sequence region to the complementary U2′ sequence region of theS-oligonucleotide which has a 3′ blocked end (⊖) and a 5′ region that ishybridized at its U1′ sequence region to a complementary U1 sequenceregion in the TSU promoter primer that includes a 5′ promoter sequenceregion (solid line P) and a 3′ target specific sequence region (TS1)which is complementary to the TS1′ sequence in the target strand. Thetarget strand also contains another target specific sequence region(TS2) that is the same as the TS2 region of the TSU non-promoter primer.The capture probe contains a 5′ target specific sequence (TS3) that iscomplementary to part of the target strand (sequence TS3′) and a 3′poly-A sequence that is complementary to a poly-T sequence that servesas the BPM of the immobilized probe.

FIG. 4 is a schematic drawing that illustrates a TSU primer complex inwhich the upper strand is a TSU non-promoter primer made up of a 3′target specific region (TS2) and a 5′ universal sequence region, labeledU2(+), which is hybridized to a complementary 3′ U2′ sequence region ofthe S-oligonucleotide (labeled S-oligo) which is contains an abasicspacer that links the 3′ U2′ sequence to a 5′ U1′ sequence region thatis the complement of and hybridized to the U1(−) sequence region in theTSU promoter primer that includes a 5′ promoter sequence (P) and a 3′target specific sequence region (TS1). The illustrated S-oligonucleotideincludes a 3′ blocked end in which terminal bases are joined by a 3′ to3′ linkage (labeled 3′-3′C) and an internal abasic compound (e.g.,(C9).sub.2 or (C9).sub.3) that is a spacer that covalently joins the 5′U1′ sequence and the 3′ U2′ sequence.

FIG. 5 is a schematic drawing that illustrates the product that resultsfrom an initial synthetic step of the initial amplification phase inwhich the 3′ end of the TSU promoter primer, hybridized via its TS1sequence to the complementary TS1′ sequence in an RNA template strand(narrow solid line), has been synthetically extended to make a firststrand cDNA (wider solid line) by using a reverse transcriptase (RT)polymerase. The RNA template strand also contains a TS2 sequence that iscomplementary to the TS2′ sequence made in the first strand cDNA.

FIG. 6 is a schematic drawing that illustrates the first strand cDNAproduct (as shown in FIG. 5) following degradation of the RNA templatestrand that was shown in FIG. 5, in which the cDNA contains a 5′promoter sequence (P), a universal sequence (U1), a target-specificsequence (TS1), a cDNA sequence that was made from the template strandand that contains a second target-specific sequence (TS2′).

FIG. 7 is a schematic drawing that illustrates the product that resultsfrom a second synthetic step in the initial phase of amplification. Thisproduct results from hybridization of the TSU non-promoter primer to thefirst strand cDNA product (see FIG. 6) by hybridizing the TS2 sequenceof the TSU non-promoter primer to the complementary TS2′ sequence of thecDNA and extending the 3′ end of the TSU non-promoter primer by using aDNA polymerase (shaded rectangle) to make a complementary second strandof DNA. The second strand contains the primer's 5′ U2 sequence and TS2sequence, the complementary sequence to the first strand cDNA whichincludes a target specific sequence TS1′, a universal sequence U1′ and a3′ sequence that is complementary to the promoter sequence of the cDNA,thus making a double-stranded DNA that contains a functional promotersequence.

FIG. 8 is a schematic drawing that illustrates the substantially dsDNAmade up of the first strand cDNA and the second strand DNA (see FIG. 7)and three RNA transcripts (broader lines) above the dsDNA. RNAtranscripts are made by transcription that initiates at the functionaldouble-stranded promoter sequence (P) by using its respective RNApolymerase (shaded area labeled RNA Pol). RNA transcripts include, in a5′ to 3′ direction, a 5′ U1 sequence, a TS1 sequence, a transcript fromthe target strand, a TS2′ sequence, and a 3′ U2′ sequence.

FIG. 9 is a schematic drawing showing a single RNA transcript, asillustrated in FIG. 8, from the first phase of isothermal amplificationwith terminal universal sequences, U1 and U2′, which flank the targetspecific sequences TS1 and TS2′, which flank the transcript of othertarget strand sequence, and a universal primer (UP2) that includessequence U2 that is complementary to sequence U2′ in the transcript.

FIG. 10 is a schematic drawing showing the steps in the second phase ofisothermal amplification in which RNA transcripts (as illustrated inFIG. 9) enter the system at the lower left where the RNA transcripthybridizes to the universal primer UP2 via complementary pairing of theU2′ and U2 sequences (hybridization shown by vertical lines ∥∥|) andreverse transcriptase enzyme (open circle labeled RT) attaches to UP2and uses its RNA directed DNA polymerase activity to enzymaticallyextend the UP2 primer by using the RNA transcript as a template. Thenext step, after the arrow pointing to the right, shows the resultingcDNA (lower strand) hybridized to the RNA template (upper strand), whichafter the upward pointing arrow, is digested by RNAse H activity of theRT enzyme that leaves the cDNA strand. After the next upward pointingarrow, the cDNA is hybridized via its U1′ sequence to the complementaryU1 sequence of the universal promoter primer (UP1) which includes a 5′promoter sequence (P) and the UP1 primer is extended by DNA directed DNApolymerase activity of the RT enzyme to make a dsDNA that is illustratedat the top of the circle, above the arrow pointing upward and leftward.The dsDNA contains two universal sequences per strand (U1 and U2′ on theupper strand and U1′ and U2 on the lower strand) that flank targetspecific sequences (TS1, TS2′ and the intervening sequence on the upperstrand and TS1′ and TS2 and the intervening sequence on the lowerstrand), and a functional promoter (P). Following the arrow downward tothe left, the functional promoter interacts with a RNA polymerase (ovallabeled RNA Pol) specific for the promoter sequence to make transcriptsfrom the dsDNA, which are shown after the next downward pointing arrow,to result in 100 to 1000 transcripts or RNA amplicons which contain twouniversal sequences (U1 and U2′) and target specific sequences (TS1 andTS2′ and the intervening sequence). Following the next arrow downwardand to the right, these RNA transcripts enter the amplification systemand are used as templates for further isothermal amplification in acyclic manner as shown, repeating the steps as described above for thefirst phase RNA transcripts.

FIG. 11 is a schematic drawing of two embodiments of TSU primers that donot include an S-oligonucleotide but which may be used in the firstphase of isothermal amplification which is performed using TSU primersattached to a support, followed by the second phase of isothermalamplification performed in solution phase by using the universal primers(UP1 and UP2). In Embodiment 1, a TSU non-promoter primer and a TSUpromoter primer are linked together, covalently or non-covalently, andattached to a support via a first binding pair member (shaded arrowlabeled BPM1) which binds specifically to a second binding pair member(dark chevron labeled BPM2) attached to the support (shaded rectangle).In Embodiment 2, the TSU non-promoter primer and TSU promoter primer areseparate oligonucleotides which are separately attached to the samesupport via a BPM1 attached to each oligomer, which binds specificallyto a separate binding pair member, BPM2, attached to the support (shadedcircle). For both Embodiment 1 and 2, universal primers (UP1 and UP2)are provided in solution phase and are unattached to a support.

FIG. 12 is a schematic drawing showing structures used in a targetcapture (TC) step with initial primer attachment (left side, labeled A.)and primers used in the second phase of isothermal amplification (rightside, labeled B.), for Embodiment 1 (upper half above the line) andEmbodiment 2 (lower half below the line). In Embodiment 1, the TC step(left side, upper half) includes a capture complex made up of the targetnucleic acid attached to a support, via a target specific capture probethat hybridizes to the target strand (shown by vertical lines between ashort horizontal line and the longer horizontal line representing thetarget strand) and also hybridizes via a poly-A sequence to animmobilize poly-T sequence attached to the support (shaded circle). Thetarget nucleic acid is attached at another location to a TSU primercomplex that includes the TSU promoter primer hybridized specifically toa sequence in the target strand and to an S-oligonucleotide that ishybridized to a TSU non-promoter primer (substantially as shown in FIG.3). In Embodiment 1, the second phase of amplification (right side,upper half) uses two universal primers: a universal promoter primer(UP1) and a universal non-promoter primer (UP2) that hybridizes to acomplementary sequence introduced in the RNA transcript by use of theTSU primer complex. In embodiment 2, the TC step (left side, lower half)includes the capture complex as shown for embodiment 1 and only the TSUpromoter primer hybridized via a target-specific sequence at anotherlocation on the target strand, and the second phase of amplification(right side, lower half) uses one universal promoter primer (UP1) andone target specific primer (TSP).

FIG. 13 is a schematic drawing showing the steps in the second phase ofisothermal amplification substantially as shown in FIG. 10, except thatRNA transcripts from the first and/or second phases (lower left) arehybridized to a target specific primer (TSP) that is extended by RT tosynthesize the cDNA strand (lower right) using the RNA transcripts astemplates, and no U2 or UT universal sequences are present.

FIG. 14 is a schematic drawing showing an embodiment in which (lowerleft) a TSU promoter primer used in a first phase of amplification isattached to a support via a first binding pair member (BPM1) that bindsspecifically to a second binding pair member (BPM2) attached to thesupport (shaded circle), and a mixture of universal promoter primers(UP1) and target specific primers (TSP) in solution phase are used inthe second phase of amplification.

FIG. 15 is a schematic drawing showing components in an embodiment inwhich the top portion of the diagram shows a hybridization complex madein the Target Capture step, made up of the Target nucleic acid strandhybridized to a target capture (TC) probe that has an unbound poly-Atail and a TS sequence hybridized to a 5′ portion of the target strand,a Blocker oligonucleotide hybridized to the target strand downstreamfrom the position hybridized to the TC probe, and a TSU primerhybridized to a 3′ portion of the target strand via a TS sequence withan unhybridized universal (U) sequence; and the lower portion of thediagram shows that the nucleic acids present in single-primer isothermalamplification which include (1) the target amplicon consisting of a 5′ Usequence, an internal TS sequence, and a 3′ sequence copied from thetarget strand by extension of the TSU primer, (2) a TS promoter providerthat includes a 5′ promoter (P) sequence, a 3′ TS sequence, and ablocked 3′ end (

), and (3) a universal primer consisting of a universal sequence (U′)complementary to the universal sequence of the target amplicon.

FIG. 16 is a schematic drawing showing components in an embodiment inwhich the top portion of the diagram shows a hybridization complex madein the Target Capture step, made up of the Target nucleic acid strandhybridized to a target capture (TC) probe that has an unbound poly-Atail and a TS sequence hybridized to a 5′ portion of the target strand,a Blocker oligonucleotide hybridized to the target strand downstreamfrom the position hybridized to the TC probe, and a TSU primer complexmade up of (top strand) a TSU promoter provider with a 3′ blocked end (

), an S-oligomer (middle strand, substantially as in FIG. 3), and a TSUprimer (lower strand) hybridized to a 3′ portion of the target strandvia a TS sequence with its universal (U2) sequence hybridized to acomplementary (U2′) sequence in the S-oligomer; and the lower portion ofthe diagram shows that nucleic acids present in single-primer isothermalamplification which include (1) the TSU promoter provider hybridized viaits TS1 sequence to the extension product made by extension of the TS2sequence of the TSU primer which includes its U2 universal sequence, (2)a promoter provider oligonucleotide that includes a 5′ promoter (P)sequence, a 3′ U1′ universal sequence, and a blocked 3′ end (

), and (3) a universal primer consisting of a universal sequence (U2′)complementary to the U2 universal sequence.

FIG. 17 is a schematic drawing of an embodiment showing two TSUoligonucleotides in a hybridization complex that is hybridized to atarget strand via the TS1 sequence of a TSU primer which also includes aU1 sequence and a promoter complementary sequence (P′), which ishybridized to a TSU promoter provider oligonucleotide via hybridizationof the complementary P′ sequence and the P sequence of the TSU promoterprovider oligonucleotide which also contains a U2 sequence, a TS2sequence and a blocked 3′ end.

FIG. 18 is a schematic drawing of an embodiment showing two TSUoligonucleotides joined covalently via a non-nucleotide linker(—C₉-C₉—). This forms a complex made up of a TSU promoter provider thatincludes a blocked 3′ end, and TS2, U2 and promoter (P) sequences in a3′ to 5′ orientation linked to a TSU primer that includes U1 and TS1sequences in a 5′ to 3′ orientation, providing one extendable 3′terminus in the complex which is hybridized to a target strand via theTS1 sequence of the TSU primer. Also shown hybridized to the Targetstrand are a blocker oligonucleotide and a TC probe, hybridized to thetarget via its TS sequence and shown with an unhybridized tail sequence.

FIG. 19 shows data obtained from an isothermal amplification of a singletarget (“PCA3 uniplex” panel) present in samples at 10.sup.2, 10.sup.4and 10.sup.6 copies per reaction, and of two targets (“PCA3/PSA duplex(oligos)” panel) present in samples at 10.sup.6 copies per reaction, inwhich amplification products were detected in real time by using afluorescent-labeled probe. For both panels, the x-axis shows cycles ofamplification and the y-axis shows fluorescence units.

DETAILED DESCRIPTION

Methods and compositions are disclosed that are useful for amplifyingtarget nucleic acid sequences in vitro in substantially isothermalconditions to produce amplified sequences that can be detected toindicate the presence of the target nucleic acid in a sample. Themethods and compositions are useful for synthesizing amplified nucleicacids to provide useful information for making diagnoses and/orprognoses of medical conditions, detecting the purity or quality ofenvironmental and/or food samples, or investigating forensic evidence.The methods and compositions are advantageous because they allowsynthesis of a variety of nucleic acids to provide highly sensitiveassays over a wide dynamic range that are relatively rapid andinexpensive to perform, making them suitable for use in high throughputand/or automated systems. The methods and compositions are useful forassays that simultaneously analyze multiple different genetic sequences,i.e., multiplex amplification systems. Preferred compositions areprovided in kits that include defined assay components that are usefulbecause they allow a user to efficiently perform methods that use thecomponents together in an assay to amplify desired targets.

The disclosed compositions and methods increase the efficiency ofisothermal amplification of nucleic acids, which is particularly usefulin multiplex assays that amplify multiple analytes in a single reactionmixture, e.g., for array-based assays. Multiplex isothermaltranscription based amplification assays are often limited toamplification of about six or fewer analyte targets in a single reactionbecause of primer/primer interactions and spurious product formation,which result in inefficient amplification of one or more of the targets,thereby decreases assay sensitivity. Although design and testing of manydifferent primers and primer combinations may improve amplificationefficiency in certain multiplex assays, the disclosed systems minimizethe common problems encountered in multiplex reactions by usingtarget-specific primers in an initial phase of amplification followed byuse of universal primers to amplify all of target amplicons in a secondphase of amplification. Thus, amplification efficiency increases whilethe need to design and test many individual primers or primercombinations in multiplex reactions decreases. That is, for each desiredtarget only one or a pair of amplification oligomer complexes unique tothe desired target are designed for use in an initial amplificationphase, and a subsequent amplification phase uses universal reagents thatare used in common for amplification of many targets. The disclosedmethods are versatile and may be used to detect a single target ormultiple different targets, all amplified in a single reaction, fromwhich amplification products may be detected at the end of a reaction(end-point detection) or during the reaction (real-time detection).Typically, the amplification oligomer primers and/or providers areprovided in a target capture reagent (TCR), preferably as anamplification oligomer complex, and so these complexed oligomers and thetarget capture oligomers are hybridized to a target nucleic acid, andisolated along with during the target capture step. Then, an initialphase of amplification is then performed. One advantage is that byhybridizing the amplification oligomer complex to the target nucleicacid during target capture, the captured nucleic acids can be washed toremove sample components, such as unhybridized amplification oligomercomplexes. In a multiplex reaction, removing unhybridized amplificationoligomers allows the multiplex amplification reaction to occur withoutinterference from these excess amplification oligomers, therebysubstantially reducing or eliminating the problems common to multiplexreactions. Further, if the amplification oligomer complex comprises oneor more members comprising a universal tag sequence, then the Usequences are incorporated into the initial amplification products,thereby allowing for subsequent amplification using universal primersspecific for these universal sequences. Furthermore, following initialmultiplex amplification, such as a pre-amplification using aamplification oligomer complex, the pre-amplified sample can be splitinto two or more secondary amplification reactions each comprisingspecific amplification oligomer for each of the targets. This multiplexpre-amplification followed by secondary target specific amplification isan advantageous use of the amplification oligomer complexes. For one,the multiplex pre-amplification uses only the amplification oligomershybridized to target nucleic acids; unhybridized amplification oligomersare removed from the reaction. This substantially reduces or eliminatesinterference in the reaction caused by the amplification oligomers forthe various targets. Pre-amplification then generates a plurality of RNAtranscripts from each of the target nucleic acid using the amplificationoligomer complex as is described herein. These RNA transcripts are thensplit into a plurality of secondary amplification reactions, each havinga single set of target specific amplification oligomers for theisothermal amplification of one of the species of RNA transcripts. Thus,in the secondary amplification reaction, there is again no interferencefrom having a plurality of amplification oligomer pairs in the reaction,as would occur with a multiplex reaction. Further, because thepre-amplification reaction had no excess oligomers, interferingoligomers are not transferred into the target specific secondaryamplification, here too avoiding the problems common in multiplexreactions, such as is the case with two-step multiplex reactions in theart.

Unless otherwise described, scientific and technical terms used hereinhave the same meaning as commonly understood by those skilled in the artof molecular biology based on technical literature, e.g., DictionaryofMicrobiology and Molecular Biology, 2nd ed. (Singleton et al., 1994,John Wiley & Sons, New York, N.Y.), or other well known technicalpublications related to molecular biology. Unless otherwise described,techniques employed or contemplated herein are standard methods wellknown in the art of molecular biology. To aid in understanding aspectsof the disclosed methods and compositions, some terms are described inmore detail or illustrated by embodiments described herein.

Nucleic acid refers to a polynucleotide compound, which includesoligonucleotides, comprising nucleosides or nucleoside analogs that havenitrogenous heterocyclic bases or base analogs, covalently linked bystandard phosphodiester bonds or other linkages. Nucleic acids includeRNA, DNA, chimeric DNA-RNA polymers or analogs thereof. In a nucleicacid, the backbone may be made up of a variety of linkages, includingone or more of sugar-phosphodiester linkages, peptide-nucleic acid (PNA)linkages (PCT No. WO 95/32305), phosphorothioate linkages,methylphosphonate linkages, or combinations thereof. Sugar moieties in anucleic acid may be ribose, deoxyribose, or similar compounds withsubstitutions, e.g., 2′ methoxy and 2′ halide (e.g., 2′-F)substitutions. Nitrogenous bases may be conventional bases (A, G, C, T,U), analogs thereof (e.g., inosine; The Biochemistry of the NucleicAcids 5-36, Adams et al., ed., 11th ed., 1992), derivatives of purine orpyrimidine bases (e.g., .sup.N4-methyl deoxygaunosine, deaza- oraza-purines, deaza- or aza-pyrimidines, pyrimidines or purines withaltered or replacement substituent groups at any of a variety ofchemical positions, e.g., 2-amino-6-methylaminopurine,O.sup.6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O.sup.4-alkyl-pyrimidines, orpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine (e.g. U.S. Pat. Nos. 5,378,825, 6,949,367 andPCT No. WO 93/13121). Nucleic acids may include “abasic” positions inwhich the backbone does not have a nitrogenous base at one or morelocations (U.S. Pat. No. 5,585,481), e.g., one or more abasic positionsmay form a linker region that joins separate oligonucleotide sequencestogether. A nucleic acid may comprise only conventional sugars, bases,and linkages as found in conventional RNA and DNA, or may includeconventional components and substitutions (e.g., conventional baseslinked by a 2′ methoxy backbone, or a polymer containing a mixture ofconventional bases and one or more analogs). The term includes “lockednucleic acids” (LNA), which contain one or more LNA nucleotide monomerswith a bicyclic furanose unit locked in a RNA mimicking sugarconformation, which enhances hybridization affinity for complementarysequences in ssRNA, ssDNA, or dsDNA (Vester et al., 2004, Biochemistry43(42):13233-41).

The interchangeable terms “oligonucleotide” and “oligomer” refer tonucleic acid polymers generally made of less than 1,000 nucleotide (nt),including those in a size range having a lower limit of about 2 to 5 ntand an upper limit of about 500 to 900 nt. Preferred oligomers are in asize range having a 5 to 15 nt lower limit and a 50 to 500 nt upperlimit, and particularly preferred embodiments are in a size range havinga 10 to 20 nt lower limit and a 25 to 150 nt upper limit Preferredoligonucleotides are made synthetically by using any well-known in vitrochemical or enzymatic method, and may be purified after synthesis byusing standard methods, e.g., high-performance liquid chromatography(HPLC). Representative oligonucleotides discussed herein include,non-promoter primers, promoter primers, promoter providers (which arepromoter primers comprising a blocked 3′-end), detection probeoligomers, target capture oligomers, and blockers, to name a few.Promoter primers, promoter providers and non-promoter primers areamplification oligomers. An amplification oligomer complex is two ofthese amplification oligomers directly or indirectly joined together, asis discussed below. Thus, an amplification oligomer complex is made of afirst amplification oligomer member and a second amplification oligomermember that are joined together. Additionally, amplification oligomerscan have universal tag sequences, as is also described herein. Theseuniversal tag nucleotide sequences are present on one or both of thefirst and second amplification oligomers. The universal tag sequencespresent on first and second amplification oligomer members can beidentical sequences, substantially identical sequences or differentsequences. Further, for multiplex amplification reactions wherein thereare two or more different first amplification oligomer members and twoor more different second amplification oligomer members, individuallyeach amplification oligomer member can have a tag sequence. If more thanone amplification oligomer member for the multiplex amplificationreaction has a tag sequence, they may be identical, substantiallyidentical, different or a combination thereof, when the tag sequencesare compared one to another. Similarly, amplification oligomer complexescan have amplification oligomer members that have tag sequences, and thetag sequences can be identical, substantially identical or different.Same for a multiplex amplification reaction wherein there are two ormore different amplification oligomer complexes, one or moreamplification oligomer members can have a tag sequences and the tagsequences can be identical, substantially identical or different whencompared one to another. Tag sequences are also referred to as “U”sequences for universal.

Oligonucleotides that are not extended enzymatically include promoterprovider oligomers and blocker oligomers. These oligomers hybridize to atarget nucleic acid, or its complement, but do not participate in an invitro nucleic acid amplification reaction wherein new nucleic acidstrands are synthesized from a template strand by using an end of thepromoter provider or blocker as an initiation point for a nucleic acidsynthesis that is catalyzed by enzymatic polymerase activity. To preventenzymatic extension of an oligonucleotide, the 3′-terminus of theoligonucleotide can be chemically or structurally blocked using ablocking moiety, as is generally known in the art. Blockedoligonucleotides are described in, e.g., U.S. Pat. Nos. 5,399,491,5,554,516, 5,824,518, and US App. No. 2006-0046265. A blockedoligonucleotide refers to an oligonucleotide that includes a chemicaland/or structural modification that is usually near or at the 3′terminus and that prevents or impedes initiation of DNA synthesis fromthe oligonucleotide by enzymatic means. Examples of such modificationsinclude use of a 3′2′-dideoxynucleotide base, a 3′ non-nucleotide moietythat prevents enzymatic extension, or attachment of a short sequence in3′ to 5′ orientation to the oligonucleotide to make a finaloligonucleotide with two 5′ termini (i.e., a first 5′ to 3′oligonucleotide attached to a second, usually shorter, 5′ to 3′oligonucleotide by covalently joining the oligonucleotides at their 3′termini). Another example of a modification is a “cap” made up of asequence that is complementary to at least 3 nt at the 3′-end of theoligonucleotide such that the 5′-terminal base of the cap iscomplementary to the 3′-terminal base of the oligonucleotide. Althoughblocked oligonucleotides are not extended synthetically, they mayparticipate in nucleic acid amplification, e.g., by hybridizing to aspecific location on a nucleic acid template strand to impede synthesisof a complementary strand beyond the position at which the blockedoligonucleotide is bound. Amplification oligonucleotides that areextended enzymatically include primers and promoter-primers. Universalprimers (UP) contain a sequence used to amplify an initial amplificationproduct (or analyte) sequence containing a universal or tag sequencethat has been incorporated into the initial amplification product.Universal primers (UP) may contain only a nucleotide sequence that issubstantially complementary to a universal sequence. A UP may furthercontain a nucleotide sequence, such as a promoter sequence. UP sequencesmay also comprise blocked 3′ termini. Terms such as “universalnon-promoter primer” “universal promoter provider” or “universalpromoter primer” may be used to distinguish between different UP types.

Sizes of the amplification oligonucleotides are generally determined bythe function portions that are included in the oligonucleotide.Component portions of a promoter primer or promoter provideroligonucleotide include a promoter sequence specific for a RNApolymerase (RNP). RNP and their corresponding promoter sequences arewell known and may be purified from or made synthetically in vitro byusing materials derived from a variety of sources, e.g., viruses,bacteriophages, fungi, yeast, bacteria, animal, plant or human cells.Examples of RNP and promoters include RNA polymerase III and itspromoter (U.S. Pat. No. 7,241,618), bacteriophage T7 RNA polymerase andits promoter or mutants thereof (U.S. Pat. Nos. 7,229,765 and7,078,170), RNA polymerase and promoter from Thermus thermophilus (U.S.Pat. No. 7,186,525), RNA polymerases from HIV-1 or HCV, and plantdirected RNPs (U.S. Pat. No. 7,060,813). A promoter primer or provideroligonucleotide includes a promoter sequence that is linked functionallyto the chosen RNP. Preferred embodiments of promoter primer or promoterprovider oligonucleotides include a T7 promoter sequence that is usedwith T7 RNP, where the promoter sequence is in the range of 25 to 30 nt,such as a promoter sequence of SEQ ID NOS. 67 or 68 (SEQ ID NO:67,aatttaatacgactcactatagggaga; SEQ ID NO:68,gaaattaatacgactcactatagggaga). Amplification oligonucleotides thatinclude a universal (U) portion typically include a U sequence in arange of 5 to 40 nt, with preferred embodiments in a range of 10 to 25nt, or 10 to 30 nt, or 15 to 30 nt. Amplification oligonucleotides thatinclude a target specific (TS) portion typically include a TS sequencein a range of 10 to 45 nt, with preferred embodiments in a range of 10to 35 nt or 20 to 30 nt. Amplification oligonucleotides that includemultiple U sequences and/or multiple TS sequences will be in a sizerange that is determined by the length of its individual functionalsequences, e.g., a promoter primer or provider oligonucleotide thatincludes a U sequence and a TS sequence will be the sum of the sizes ofthe promoter, U and TS sequences, and may optionally include linkingnucleotides or non-nucleotide portions (e.g., abasic linkers).Amplification oligonucleotides made up of multiple functional componentsas described herein may be covalently linked by standard phosphodiesterlinkages, nucleic acid analog linkages, or non-nucleic acid linkagesdirectly between the different functional portions or may be covalentlylinked together by using additional nucleic acid sequences ornon-nucleic (e.g., abasic linkages) compounds that serve as spacersbetween functional portions. Some embodiments of amplificationoligonucleotides may be linked together to form a complex by usingnon-covalent linkages, such as by using interactions of binding pairmembers between the oligonucleotides, which includes directhybridization of complementary sequences contained in two or moreoligonucleotides, or via a linking component to which the individualbinding pair member of an oligonucleotide binds (e.g., a binding pairmember for each oligonucleotide attached to a support).

A promoter provider oligonucleotide refers to an oligonucleotide thatcontains a promoter sequence usually on an oligonucleotide that includesa first region that hybridizes to a 3′-region of a DNA primer extensionproduct (e.g., a cDNA) to form a hybridization complex between thepromoter provider oligonucleotide and the extension product, and asecond region, located 5′ to the first region, that is a promotersequence for an RNA polymerase. By forming the hybridization complexwith the extension product, the promoter provider oligonucleotide canserve as a template for making a dsDNA that includes a functionalpromoter when the extension product or cDNA is used as a template forfurther strand synthesis, i.e., by extending a newly synthesized strandmade from using the cDNA as a template and using the promoter sequenceof the promoter provider oligonucleotide as a template, a substantiallydouble-stranded structure that contains a functional promoter issynthesized in vitro.

Amplification of a nucleic acid refers to the process of creating invitro nucleic acid strands that are identical or complementary to acomplete or portion of a target nucleic acid sequence, or a universal ortag sequence that serves as a surrogate for the target nucleic acidsequence, all of which are only made if the target nucleic acid ispresent in a sample. Typically, nucleic acid amplification uses one ormore nucleic acid polymerase and/or transcriptase enzymes to producemultiple copies of a target polynucleotide or fragments thereof, or of asequence complementary to the target polynucleotide or fragmentsthereof, or of a universal or tag sequence that has been introduced intothe amplification system to serve as a surrogate for the targetpolynucleotide, such as in a detection step, to indicate the presence ofthe target polynucleotide at some point in the assay. In vitro nucleicacid amplification techniques are well known and includetranscription-associated amplification methods, such as transcriptionmediated amplification (TMA) or nucleic acid sequence basedamplification (NASBA), and other methods such as the Polymerase ChainReaction (PCR), reverse transcriptase-PCR, replicase mediatedamplification, and the Ligase Chain Reaction (LCR).

To aid in understanding some of the embodiments disclosed herein, theTMA method that has been described in detail previously (e.g., U.S. Pat.Nos. 5,399,491, 5,554,516 and 5,824,518) is briefly summarized. In TMA,a target nucleic acid that contains the sequence to be amplified isprovided as single stranded nucleic acid (e.g., ssRNA or ssDNA). Anyconventional method of converting a double stranded nucleic acid (e.g.,dsDNA) to a single-stranded nucleic acid may be used. A promoter primerbinds specifically to the target nucleic acid at its target sequence anda reverse transcriptase (RT) extends the 3′ end of the promoter primerusing the target strand as a template to create a cDNA copy, resultingin a RNA:cDNA duplex. RNase activity (e.g., RNaseH of RT enzyme) digeststhe RNA of the RNA:cDNA duplex and a second primer binds specifically toits target sequence in the cDNA, downstream from the promoter-primerend. Then RT synthesizes a new DNA strand by extending the 3′ end of thesecond primer using the cDNA as a template to create a dsDNA thatcontains a functional promoter sequence. RNA polymerase specific for thefunctional promoter initiates transcription to produce about 100 to 1000RNA transcripts (amplified copies or amplicons) of the initial targetstrand. The second primer binds specifically to its target sequence ineach amplicon and RT creates a cDNA from the amplicon RNA template toproduce a RNA:cDNA duplex. RNase digests the amplicon RNA from theRNA:cDNA duplex and the target-specific sequence of the promoter primerbinds to its complementary sequence in the newly synthesized DNA and RTextends the 3′ end of the promoter primer to create a dsDNA thatcontains a functional promoter to which the RNA polymerase binds andtranscribes additional amplicons that are complementary to the targetstrand. Autocatalytic cycles that use these steps repeatedly during thereaction produce about a billion-fold amplification of the initialtarget sequence. Amplicons may be detected during amplification(real-time detection) or at an end point of the reaction (end-pointdetection) by using a probe that binds specifically to a sequencecontained in the amplicons. Detection of a signal resulting from thebound probes indicates the presence of the target nucleic acid in thesample.

Another form of transcription associated amplification that uses asingle primer and one or more additional amplification oligomers toamplify nucleic acids in vitro by making transcripts that indicate thepresence of the target nucleic acid has been described in detailpreviously (US App. 2006-0046265). Briefly, this single-primer methoduses a priming oligomer, a promoter oligomer (or promoter provideroligonucleotide) that is modified to prevent the initiation of DNAsynthesis from its 3′ end and, optionally, a binding molecule (e.g., a3′-blocked oligomer) to terminate elongation of a cDNA from the targetstrand. The method synthesizes multiple copies of a target sequence bytreating a target nucleic acid that includes a RNA target sequence with(i) a priming oligonucleotide which hybridizes to the 3′-end of thetarget sequence such that a primer extension reaction can be initiatedtherefrom and (ii) a binding molecule that binds to the target nucleicacid adjacent to or near the 5′-end of the target sequence. The primingoligonucleotide is extended in a primer extension reaction by using aDNA polymerase to give a DNA primer extension product complementary tothe target sequence, in which the DNA primer extension product has a 3′end determined by the binding molecule and which is complementary to the5′-end of the target sequence. The method then separates the DNA primerextension product from the target sequence by using an enzyme whichselectively degrades the target sequence and treats the DNA primerextension product with a promoter oligonucleotide made up of a firstregion that hybridizes to a 3′-region of the DNA primer extensionproduct to form a promoter oligonucleotide:DNA primer extension producthybrid, and a second region that is a promoter for an RNA polymerasewhich is situated 5′ to the first region, wherein the promoteroligonucleotide is modified to prevent the initiation of DNA synthesisfrom the promoter oligonucleotide. The method extends the 3′-end of theDNA primer extension product in the promoter oligonucleotide:DNA primerextension product hybrid to add a sequence complementary to the secondregion of the promoter oligonucleotide, which is used to transcribemultiple RNA products complementary to the DNA primer extension productusing an RNA polymerase which recognizes the promoter and initiatestranscription therefrom. This method produces RNA transcripts that aresubstantially identical to the target sequence.

An embodiment of the one-primer transcription mediated amplificationmethod synthesizes multiple copies of an RNA target sequence byhybridizing to the target RNA a primer at a location in the 3′ portionof the target sequence and a 3′ blocked oligomer (i.e., the blockeroligomer) at a location in the 5′ portion of the target sequence. Thenthe DNA polymerase activity of RT initiates extensions from the 3′ endof the primer to produce a cDNA in a duplex with the template strand (aRNA:cDNA duplex). The 3′ blocked oligomer binds to the target strand ata position adjacent to the intended 5′ end of the sequence to beamplified because the bound 3′ blocked oligomer impedes extension of thecDNA beyond that location. That is, the 3′ end of the cDNA is determinedby the position of the binding molecule because polymerization stopswhen the extension product reaches the blocking molecule bound to thetarget strand. The RNA:cDNA duplex is separated by RNAse activity (RNaseH of RT) that degrades the RNA, although those skilled in the art willappreciate that any form of strand separation may be used. A promoterprovider oligomer includes a 5′ promoter sequence for an RNA polymeraseand a 3′ sequence complementary to a sequence in the 3′ region of thecDNA to which it hybridizes. The promoter provider oligomer has amodified 3′ end that includes a blocking moiety to prevent initiation ofDNA synthesis from the 3′ end of the promoter provider oligomer. In theduplex made of the promoter provider hybridized to the cDNA, the 3′-endof the cDNA is extended by using DNA polymerase activity of RT and thepromoter provider oligomer serves as a template to add a promotersequence to the 3′ end of the cDNA, which creates a functionaldouble-stranded promoter made up of the sequence on the promoterprovider oligomer and the complementary cDNA sequence made from thepromoter provider template. RNA polymerase specific for the promotersequence binds to the functional promoter and transcribes multiple RNAtranscripts that are complementary to the cDNA and substantiallyidentical to the target sequence of the initial target RNA strand. Theresulting amplified RNA can cycle through the process again by bindingthe primer and serving as a template for further cDNA production,ultimately producing many amplicons from the initial target nucleic acidpresent in the sample. Embodiments of the single primer transcriptionassociated amplification method do not require use of the 3′ blockedoligomer that serves as a binding molecule and, if a binding molecule isnot included the cDNA product made from the primer has an indeterminate3′ end, but amplification proceeds substantially the same as describedabove. Due to the nature of this amplification method, it is performedunder substantially isothermal conditions, i.e., without cycles ofraising and lowering incubation temperatures to separate strands orallow hybridization of primers as is used in PCR-based methods.

Detection of the amplified products may be accomplished by using anyknown method. For example, the amplified nucleic acids may be associatedwith a surface that results in a detectable physical change, e.g., anelectrical change. Amplified nucleic acids may be detected in solutionphase or by concentrating them in or on a matrix and detecting labelsassociated with them (e.g., an intercalating agent such as ethidiumbromide or cyber green). Other detection methods use probescomplementary to a sequence in the amplified product and detect thepresence of the probe:product complex, or use a complex of probes toamplify the signal detected from amplified products (e.g., U.S. Pat.Nos. 5,424,413, 5,451,503 and U.S. Pat. No. 5,849,481). Other detectionmethods use a probe in which signal production is linked to the presenceof the target sequence because a change in signal results only when thelabeled probe binds to amplified product, such as in a molecular beacon,molecular torch, or hybridization switch probe (e.g., U.S. Pat. Nos.5,118,801, 5,312,728, 5,925,517, 6,150,097, 6,849,412, 6,835,542,6,534,274, and 6,361,945 and US Apps. 2006-0068417 A1 and US2006-0194240 A1). Such probes typically use a label (e.g., fluorophore)attached to one end of the probe and an interacting compound (e.g.,quencher) attached to another location of the probe to inhibit signalproduction from the label when the probe is in one conformation(“closed”) that indicates it is not hybridized to amplified product, buta detectable signal is produced when the probe is hybridized to theamplified product which changes its conformation (to “open”). Detectionof a signal from directly or indirectly labeled probes that specificallyassociate with the amplified product indicates the presence of thetarget nucleic acid that was amplified.

Members of a specific binding pair (or binding partners) are moietiesthat specifically recognize and bind each other. Members may be referredto as a first binding pair member (BPM1) and second binding pair member(BPM2), which represent a variety of moieties that specifically bindtogether. Specific binding pairs are exemplified by a receptor and itsligand, enzyme and its substrate, cofactor or coenzyme, an antibody orFab fragment and its antigen or ligand, a sugar and lectin, biotin andstreptavidin or avidin, a ligand and chelating agent, a protein or aminoacid and its specific binding metal such as histidine and nickel,substantially complementary polynucleotide sequences, which includecompletely or partially complementary sequences, and complementaryhomopolymeric sequences. Specific binding pairs may be naturallyoccurring (e.g., enzyme and substrate), synthetic (e.g., syntheticreceptor and synthetic ligand), or a combination of a naturallyoccurring BPM and a synthetic BPM.

Target capture refers to selectively separating a target nucleic acidfrom other components of a sample mixture, such as cellular fragments,organelles, proteins, lipids, carbohydrates, or other nucleic acids. Atarget capture system may be specific and selectively separate apredetermined target nucleic acid from other sample components, e.g., byusing a sequence specific to the intended target nucleic acid, or it maybe nonspecific and selectively separate a target nucleic acid from othersample components by using other characteristics of the target, e.g., aphysical trait of the target nucleic acid that distinguishes it fromother sample components which do not exhibit that physicalcharacteristic. Preferred target capture methods and compositions havebeen previously described in detail (U.S. Pat. Nos. 6,110,678 and6,534,273 and US App. 2008-0286775 A1). Preferred target captureembodiments use a capture probe in solution phase and an immobilizedprobe attached to a support to form a complex with the target nucleicacid and separate the captured target from other components.

A capture probe refers to at least one nucleic acid oligomer that joinsa target nucleic acid and an immobilized probe by using binding pairmembers that may be complementary nucleic acid sequences. One captureprobe embodiment binds nonspecifically to a target nucleic acid andlinks it to a support for separation from the sample, whereas anotherembodiment includes a target specific (TS) sequence that bindsspecifically to a sequence in the target nucleic acid and an immobilizedprobe-binding region that binds to an immobilized probe, e.g., byspecific binding pair interaction. In embodiments in which the TSsequence and immobilized probe-binding region are both nucleic acidsequences, they may be covalently joined or may be on differentoligonucleotides joined by one or more linkers Immobilized probe refersto a moiety attached to a support that joins the capture probe to asupport, directly or indirectly, e.g., by joining members of a specificbinding pair, which includes non-nucleic acid binding (e.g., avidin withbiotin) and nucleic acid sequence hybridization Immobilized probesinclude an oligonucleotide attached to a support to facilitateseparation of bound target from unbound material, such as other samplecomponents and/or other oligonucleotides included in a target capturereaction mixture. A target capture (TC) complex includes the captureprobe's TS sequence hybridized specifically to a sequence in the targetnucleic acid and the capture probe's immobilized probe-binding regionbound to an immobilized probe on a support.

Support refers to known materials, such as matrices or particlesdispersed in solution, which may be made of nitrocellulose, nylon,glass, polyacrylate, mixed polymers, polystyrene, silane, metal orpolypropylene. Preferred supports are magnetically attractableparticles, e.g., monodisperse magnetic spheres of uniform size ±5% toprovide consistent results, to which an immobilized probe is joineddirectly (via covalent linkage, chelation, or ionic interaction), orindirectly (via one or more linkers), to provide stable attachment ofthe immobilized probe to the support in conditions used in the targetcapture reaction. Commonly, these are referred to as magnetic beads ormagbeads.

Separating or purifying refers to removal of one or more components of amixture, such as a sample, from one or more other components in themixture. Sample components include nucleic acids in a generally aqueoussolution phase, which may include cellular fragments, proteins,carbohydrates, lipids, and other compounds. Preferred embodimentsseparate or remove at least 70% to 80%, and more preferably about 95%,of the target nucleic acid from other components in the mixture.

Label refers to a molecular moiety or compound that can be detected orlead to a detectable response, which may be joined directly orindirectly to a nucleic acid probe. Direct labeling may use bonds orinteractions to link label and probe, which includes covalent bonds,non-covalent interactions (hydrogen bonds, hydrophobic and ionicinteractions), or chelates or coordination complexes. Indirect labelingmay use a bridging moiety or linker (e.g. antibody, oligomer, or othercompound), which is directly or indirectly labeled, which may amplify asignal Labels include any detectable moiety, e.g., radionuclide, ligandsuch as biotin or avidin, enzyme, enzyme substrate, reactive group,chromophore (detectable dye, particle, or bead), fluorophore, orluminescent compound (bioluminescent, phosphorescent, orchemiluminescent label). Preferred chemiluminescent labels includeacridinium ester (“AE”) and derivatives thereof (U.S. Pat. Nos.5,656,207, 5,658,737, and 5,639,604). Preferred labels are detectable ina homogeneous assay in which bound labeled probe in a mixture exhibits adetectable change compared to that of unbound labeled probe, e.g.,stability or differential degradation, without requiring physicalseparation of bound from unbound forms (e.g., U.S. Pat. Nos. 5,283,174,5,656,207 and 5,658,737). Methods of synthesizing labels, attachinglabels to nucleic acids, and detecting labels are well known (e.g.,Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), Chapt.10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and4,581,333).

An array refers to multiple components arranged in a two-dimensional orthree-dimensional format to allow similar or identical method steps tobe performed on the components substantially simultaneously. Examples ofarrays are well known and include high-density microarrays or gene chipsthat contain 10 to thousands of oligonucleotides attached to a supportin predetermined configuration. Such arrays allow performance of assaysteps on all the oligonucleotides in different positions under the sameconditions, e.g., hybridization of nucleic acids in a sample applied tothe array or detection of specific sequences.

Sample refers to a specimen that may contain an analyte of interest,e.g., microbe, virus, nucleic acid such as a gene, or componentsthereof, which includes nucleic acid sequences in or derived from ananalyte. Samples may be from any source, such as biological specimens orenvironmental sources. Biological specimens include any tissue ormaterial derived from a living or dead organism that may contain ananalyte or nucleic acid in or derived from an analyte. Examples ofbiological samples include respiratory tissue, exudates (e.g.,bronchoalveolar lavage), biopsy, sputum, peripheral blood, plasma,serum, lymph node, gastrointestinal tissue, feces, urine, or otherfluids, tissues or materials. Examples of environmental samples includewater, ice, soil, slurries, debris, biofilms, airborne particles, andaerosols. Samples may be processed specimens or materials, such asobtained from treating a sample by using filtration, centrifugation,sedimentation, or adherence to a medium, such as matrix or support.Other processing of samples may include treatments to physically ormechanically disrupt tissue, cellular aggregates, or cells to releaseintracellular components that include nucleic acids into a solutionwhich may contain other components, such as enzymes, buffers, salts,detergents and the like.

“Consisting essentially of” is used to mean that additionalcomponent(s), composition(s) or method step(s) that do not materiallychange the basic and novel characteristics of a TSU complex compositionor an amplification method that uses universal sequences and TSsequences as described herein may be included in the compositions ormethods. Such characteristics include the structures of TSUoligonucleotides, including complexes of multiple TSU oligonucleotidesas described herein and the ability of the methods to detect one or moreanalytes or target nucleic acids in a sample by associating one or moreuniversal sequences with the respective target sequences, amplifying inan in vitro condition at least one universal sequence, and detecting aresponse resulting from amplification of the universal sequence toindicate the presence of at least one analyte in the assayed sample. Anycomponent(s), composition(s), or method step(s) that have a materialeffect on the basic characteristics of the claimed compositions and/ormethods fall outside of this term.

Preferred embodiments of the disclosed methods use aspects of isothermalamplification systems that are generally referred to as transcriptionassociated amplification methods, which have been previously describedin detail (U.S. Pat. Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238,4,868,105 and 5,124,246, PCT Nos. WO 88/01302 WO 95/03430, and WO88/10315, and US App. 2006-0046265 A1). Examples include transcriptionmediated amplification (TMA) and nucleic acid sequence basedamplification (NASBA). Typically, transcription-associated amplificationuses an RNA polymerase to produce multiple RNA transcripts from anucleic acid template by using a series of steps that employ an RNApolymerase, a DNA polymerase, deoxyribonucleoside triphosphates,ribonucleoside triphosphates, a template complementary amplificationoligonucleotide that includes a promoter sequence, and optionally one ormore other oligonucleotides, which may serve as primers. Preferreddisclosed embodiments are based on TMA (U.S. Pat. Nos. 5,399,491 and5,554,516) or one-primer transcription-associated amplification (US2006-0046265 A1), although a person of ordinary skill in the art willunderstand that other amplification methods based on polymerase mediatedextension of oligonucleotide sequences may be used with the compositionsand/or method steps described herein.

TMA methods disclosed herein use three basic steps in atranscription-associated amplification reaction. First, a target capture(TC) step includes hybridizing one or more amplification oligomers(which may be in a linked complex and which may include a universal tagsequence) to the target nucleic acid and capturing the hybridizationcomplex that includes the target and the primer(s) from a mixture thatseparates the target nucleic acid from other sample components. A targetcapture mixture may include multiple primers, each type specific for adifferent target nucleic acid that may be present in a sample mixture.During the TC step, only those primers that are specific for a targetnucleic acid that is present in the sample mixture will be bound to atarget and carried into the subsequent amplification steps, becauseprimers specific for other targets that are not present in the samplewill remain in solution phase and be discarded or washed away with othersample components before amplification begins using the captured targetnucleic acids. Thus, extraneous oligonucleotides that might otherwiseresult in interference or competition for resources during amplificationare eliminated before the amplification steps begin. The capturedtarget-primer complex is used in an isothermal amplification reaction,which is described as a first phase of amplification (or initialamplification) and a second phase of amplification (or exponentialamplification). In the first phase of amplification, an initiation stepextends the primer attached to the target nucleic acid strand byenzymatic in vitro nucleic acid synthesis which, in some embodiments,links a universal sequence region to an initial amplicon made from thetarget strand which serves as a template. For example, if the targetstrand is RNA, a TSU primer hybridizes to the RNA and serves as aninitiation site for synthesis of the cDNA strand that includes the Usequence present on the TSU primer. In the second phase ofamplification, subsequent synthetic steps in the reaction use theinitial amplicons, which may include the U sequence incorporated intothe product in the initial phase, and amplify the initial and subsequentamplicons by using target specific primer or universal primers thathybridize to the universal sequences and are extended enzymatically byusing amplicons as templates. In some embodiments, two universalsequences are introduced into the initial amplified products of theisothermal amplification reaction and those universal sequences are thetargets of subsequent amplifications that use primers that containcomplementary universal sequences to make more amplicons from thecaptured target sequence. In other embodiments, no universal sequencesare introduced into the initial amplified products of the isothermalamplification reaction and subsequent amplifications use primers thatcontain target specific sequences to make more amplicons from thecaptured target sequence. In other embodiments, one universal sequenceis introduced into the initial amplified products and in the secondamplification phase steps, primers include one with a universal sequencespecific for the introduced universal sequence and another targetspecific primer (TSP) that is specific for a sequence contained in thetarget nucleic acid strand or a complementary strand. In someembodiments, universal primers are provided in a reagent that is mixedwith the captured hybridization complexes that include the target strandand TSU primer, in which the reagent also provides one or more othercomponents used in in vitro nucleic acid synthesis (e.g., nucleotidetriphosphates, enzymes, cofactors and the like) in the second phase.

Oligonucleotides are disclosed for use in preferred embodiments of theuniversal transcription associated amplification methods that include:(1) a target specific capture oligomer (which may be referred to as acapture probe), (2) a target-specific universal (TSU) promoter primer orTSU promoter provider, (3) a target-specific universal (TSU)non-promoter primer, (4) an optional linker oligonucleotide that may bereferred to as an S-oligonucleotide which serves to link TSU primers ina complex that is hybridized via a portion of one TSU oligonucleotide tothe target strand, (5) a universal promoter primer (which may bereferred to as UP1), and (6) a universal non-promoter primer (which maybe referred to as UP2). In some embodiments, two TSU primers are linkedtogether into a complex that is then hybridized to a target strand byusing hybridization of a TS sequence in a TSU primer to a complementarysequence on the target strand. Such linking of TSU primers may bemediated by hybridization of the TSU primers to a linkingoligonucleotide, which is sometimes referred to as an S-oligonucleotidedue to its serpentine shape when it non-covalently joins two TSU primersin a three-oligonucleotide complex, in which a first end sequence of theS-oligonucleotide that is complementary to and hybridized to part of afirst TSU primer and a second end sequence of the S-oligonucleotide iscomplementary to and hybridized to part of a second TSU primer. In someembodiments, a TSU promoter primer sequence may be linked to a TSUnon-promoter primer sequence without use of a S-oligonucleotide linker.For example, a TSU promoter primer sequence and TSU non-promoter primersequence may be synthesized as a single oligonucleotide in which bothfunctional sequences are covalently linked, either directly orindirectly, such as by using an intervening spacer oligonucleotidesequence or a non-nucleotide covalent linker compound. In otherembodiments, the two TSU oligonucleotide sequences may be synthesized asseparate oligonucleotides that are joined covalently by subsequentlyligating then together directly or indirectly, e.g., by use of a randomlinker sequence. In embodiments in which multiple TSU oligonucleotidesare linked non-covalently into a complex they may be synthesized asseparate oligonucleotides and then joined to a single support, e.g., viabinding pair members attached to the support, or the separate TSUoligonucleotides may contain complementary sequences that are directlyhybridized to link the two functional TSU oligonucleotides into acomplex. For example (shown below in “Embodiment a”), a first TSUoligonucleotide is synthesized to contain, in a 5′ to 3′ orientation, a5′ promoter sequence (P), a middle universal sequence (U1), and a 3′target specific sequence (TS1), and a second TSU oligonucleotide issynthesized to contain a 5′ sequence complementary to the promotersequence (P′), a middle universal sequence (U2), and a 3′ targetspecific sequence (TS2). Alternatively (shown below in “Embodiment b”),the second TSU oligonucleotide may be without the U2 sequence to containa 5′ sequence complementary to the promoter sequence (P′) and a 3′target specific sequence (TS2). When the two TSU oligonucleotides aremixed under hybridization conditions, they form a directly hybridized(DH) complex of TSU oligonucleotides as diagrammed below, where verticallines (∥|) indicate the hybridization of the complementary P and P′sequences.

A version of Embodiment a is illustrated schematically in FIG. 17 inwhich the two TSU oligonucleotides are shown in a hybridization complexthat is hybridized to a target strand via the TS1 sequence of a firstTSU primer which is hybridized via the complementary P′ and P sequencesto the second TSU oligonucleotide, which is a TSU promoter provideroligonucleotide with a blocked 3′ end. Amplification oligomer complexesthat do not include universal sequence regions are shown below inembodiment C. Embodiment c illustrated a DH-complex, wherein P is apromoter sequence and P′ is a complementary promoter sequence. Directhybridization is not limited to promoter-complementary promoter, as isillustrated herein.

Alternatively, two primers may be linked together covalently into acomplex that is then hybridized to a target strand by usinghybridization of a TS sequence in a primer to a complementary sequenceon the target strand. FIG. 18 illustrates such an embodiment. Thisembodiment shows two TSU oligonucleotides joined covalently via anon-nucleotide linker (—C.sub.9-C.sub.9-) to form a complex made up of aTSU promoter provider that includes a blocked 3′ end, and TS2, U2 andpromoter (P) sequences in a 3′ to 5′ orientation linked to a TSU primerthat includes U1 and TS1 sequences in a 5′ to 3′ orientation. Thiscomplex provides one extendable 3′ terminus in the complex thathybridizes to a target strand via the TS1 sequence of the TSU primer.FIG. 18 also shows, hybridized to the target, a blocker oligonucleotideand a TC probe, hybridized to the target via its TS sequence. Manymethods of making covalently linked primers to make a TSU primer complexare envisioned. For example, coupling after the 2 different oligos(primer and promoter primer or provider) are synthesized by using analdehyde:hydrazine coupling pair. Other coupling pairs may be used, e.g.a carboxyl and an amine, condensed using standard carbodiimidechemistry. Alternatives for making covalently linked TSU primercomplexes include constructing the entire complex on the DNAsynthesizer. For example, by using standard 3′ to 5′ synthesis of a TSUprimer, incorporation of spacers (e.g., non-nucleotide linkers ornucleotide linkers, such as poly-T), 5′ to 3′ synthesis of the TSUpromoter primer or provider oligonucleotide by using reverse polarityphosphoramidites, and finishing the synthesis by adding a 3′ blockerstructure, e.g., a C added in 3′ to 5′ orientation. Other alternativesuse the same basic strategy, but start with the TSU T7 promoter primeror provider oligonucleotide and end with the non-promoter TSU primer.

Embodiments of the amplification oligonucleotides may be used in methodsteps in which the TSU oligonucleotides do not form a hybridizationcomplex or covalently linked complex of multiple functional sequenceregions. That is, amplification oligonucleotides may be provided insolution phase as individual oligonucleotides or mixtures ofoligonucleotides in which the individual amplification oligonucleotidesfunction in the method steps without first forming a complex of multipleamplification oligonucleotides independent of the target nucleic acid.

In some embodiments, only one TSU oligonucleotide is used in the initialamplification phase in combination with a target specific primer (TSP)that does not contain a universal (U) sequence. For example, a TSUpromoter primer or TSU promoter provider oligonucleotide may be used incombination with a TS primer, or in another example, a TSU primer may beused in combination with a promoter primer or promoter provideroligonucleotide that does not contain a U sequence. That is, only oneTSU oligonucleotide is used in the initial amplification phase tointroduce a U sequence into an amplicon made during in the initial phaseand a TS primer is used as an initiation point for enzymatic synthesisof the initial complementary strand made from the target strand or toserve as a primer to make a strand complementary to the strand made fromthe target strand. In an embodiment that uses only one TSUoligonucleotide, one universal primer specific for the universalsequence introduced by the TSU oligonucleotide is used in the secondphase of amplification. That is, a single universal sequence serves asthe surrogate or tag sequence for that target during the second phase ofamplification.

In certain embodiments in which the promoter sequence in a TSU promoterprimer or promoter provider oligonucleotide is one recognized by abacteriophage T7 RNA polymerase, the TSU promoter primer or provider maybe referred to as a “TSU T7 primer” or “TSU T7 provider” oligonucleotidewhich may be distinguished from a TSU non-promoter primeroligonucleotide (referred to as a “TSU non-T7 primer”), and a universalprimer (UP1) that includes a T7 promoter sequence may be referred to as“T7-UP1 primer” which is distinguished from a universal primer (UP2)that does not contain a promoter sequence (referred to as a “non-T7-UP2primer”).

Embodiments of compositions and steps included in amplification methodsdescribed herein are illustrated by the figures.

Referring to FIG. 1, oligonucleotides used in methods disclosed hereinare schematically drawn. At the top, a hybridization complex isillustrated that is made up of a TSU promoter primer linkednon-covalently to the S-oligonucleotide which is linked non-covalentlyto the TSU non-promoter primer. In this complex, the TSU promoter primeris diagramed at the top as including a 5′ promoter sequence (P, solidline), a middle universal sequence, U1 (dashed line), and a 3′target-specific sequence, TS1 (double line). The S-oligonucleotide isshown as an S-shaped curve (dotted line) having a 5′ region thatincludes sequence U1′ that is complementary to the universal sequence U1of the TSU promoter primer and a 3′ region that includes sequence UTthat is complementary to the universal sequence U2 of the TSUnon-promoter primer. The TSU non-promoter primer is diagramed at thebottom of the complex includes a 5′ universal sequence, U2 (dashed line)and a 3′ target-specific sequence, TS2 (double line). Hybridizationbetween the universal sequences of the TSU primers and the complementarysequences of the S-oligonucleotide forms the complex. Under the complexthat contains the TSU primers is shown the target-specific captureoligonucleotide, which is diagramed as having a 5′ target-specificregion, TS3 (double line), and a 3′ moiety that is a member of aspecific binding pair (triple line), which in some embodiments is ahomopolymeric nucleic acid sequence. Next is shown the universalpromoter primer (UP1), which is diagramed as having a 5′ promotersequence region (solid line) and a 3′ universal sequence region, U1(dashed line). Next is a diagram of the universal non-promoter primer(UP2), which is shown as a universal sequence, U2 (dashed line).

In preferred embodiments, target capture and amplificationoligonucleotides are provided in a minimum of reagents, to minimize thenumber of addition steps required to perform an assay. In one preferredembodiment, two reagent mixtures are provided as follows. A firstreagent mixture, referred to as a Target Capture Reagent (TCR),comprises the TSU primers (e.g., TSU-T7 primer and TSU non-T7 primer)and all cofactors needed for their specific attachment to the desiredtarget sequences are included (e.g., appropriate salts and buffers forhybridization when mixed with a sample that contains the target nucleicacids). The TCR also includes all of the oligonucleotides used in thetarget capture step, e.g., a capture probe specific for each desiredtarget or a non-specific capture probe, a support to capture the captureprobe attached to the target nucleic acid, and any intermediaryoligonucleotides used in target capture, such as an immobilized probe onthe support. A second reagent mixture, referred to as an AmplificationReagent (AR), provides only one set of universal primers, the universalpromoter primer and the universal non-promoter primer, in addition tocompounds used in in vitro nucleic acid synthesis, e.g., nucleotidetriphosphates (NTPs, dNTPs), salts, buffering agents, enzyme cofactors,and enzyme(s). In another preferred embodiment, a first reagent mixture(e.g., TCR) comprises TSU primers and providers (e.g., TSU T7 providersand TSU non-T7 primers) as well as needed cofactors. Target captureoligomers are also preferably included in the TCR. A second reagent(e.g., AR) comprises universal primers and providers, as well asadditional components. In another embodiment useful with amplificationreactions such as PCR, a first reagent mixture (e.g., TCR) comprises TSUprimers (e.g., two separate TSU non-T7 primers) as well as neededcofactors. Target capture oligomers are also preferably included in theTCR. A second reagent (e.g., AR) comprises universal primers, as well asadditional components. In alternate embodiments, second reagents cancomprise one universal primer or provider and one target specific primeror provider.

In use, the TCR is mixed with a sample that contains the intended targetnucleic acids. The TCR that contains target capture oligonucleotides andTSU primers, or primer and provider allows all of the introducedoligonucleotides to simultaneously hybridize specifically to theirrespective complementary sequences for each intended target nucleic acidin the sample. By including the TSU primers, or primer and provider andthe target capture oligonucleotides in the first reagent, which is mixedwith the sample, the TSU complex and the capture oligonucleotidehybridized to separate sequence regions of the target nucleic acid. Thenthe target capture oligomer is attached to a support using first andsecond binding members and is then separated from other samplecomponents, including TSU complexes that are not bound to their intendedtarget nucleic acid, thus limiting the nucleic acids carried into theamplification step to the desired targets which are already linked totheir specific TSU primers. An initial amplification product can then besynthesized using the TSU member of the TSU complex that is hybridizedto the target nucleic acid, and thus the universal sequence of that TSUmember is incorporated into a strand of the initial amplificationproduct. The second member of the TSU complex then hybridizes to itstarget sequence on the first strands of the initial amplificationproduct and a second strand can be synthesized, thereby incorporatingthe universal sequence of that TSU member into a second strand of theinitial amplification product. Preferably, the first and second TSUmembers of the TSU complex remain linked into the complex duringsynthesis of the first and second strands. By remaining linked, reactionefficiencies are increased because of the proximity of the second TSUmember to its target binding sequence, once formed. Amplification of theinitial amplification product is then performed using universalamplification oligomers. In embodiment wherein only one of the TSUmembers of the TSU complex comprises a universal sequence that getsincorporated into the initial amplification product, then secondaryamplification uses a universal amplification oligomer and a targetspecific amplification oligomer. Preferred amplification methods includeTMA, PCR and other known amplification methods.

An embodiment diagramed in FIG. 2 illustrates the target capture phaseof the universal isothermal amplification method that involves specificbinding of a target nucleic acid in the sample to its respective TSUprimers and to its respective target-specific capture oligonucleotide.FIG. 2, 1. illustrates a target capture reagent (TCR) that is a mixtureof multiple different TSU primer complexes (each containing targetspecific sequences, TSa, TSb, and TSc, which are specific for thedifferent targets, a, b, and c). The TCR also contains thetarget-specific capture oligonucleotides for each of the potentialtargets, with the 3′ member of the binding pair shown as a poly-Asequence. The TSU primer complexes are shown as a TSU promoter primerlinked via an S-oligonucleotide to a TSU non-promoter primer, and thecapture oligomers are shown as a solid line and a poly-A region, bothsubstantially as shown in FIG. 1. For each set of TSU primer complexesand capture oligomers specific for a target nucleic acid, thetarget-specific regions are labeled as TSa, TSb, or TSc. The TCR alsocontains a support with an attached immobilized moiety that bindsspecifically to the capture oligomers (see FIG. 2, 3.). In FIG. 2, 2.,the sample which contains a target nucleic acid (Target a) is mixed withthe TCR, which allows binding of the target specific sequence of the TSacapture probe to bind to its complementary sequence in Target a, and thetarget specific sequence of the promoter primer in the TSU primercomplex to bind to its complementary sequence in Target a. The poly-Asequence of the TSa capture probe binds to its complementary poly-Tsequence of the immobilized probe attached to the support, which allowsthe captured Target a with the TSa TSU primer complex to be retrievedfrom the mixture with the support (see FIG. 2, 3.). The waste productsof the target capture step, following separation of the immobilizedcomplexes on the supports, include the unbound TSU primer complexes(TSUb and TSUc primer complexes, see FIG. 2. 4.), thereby removing themfrom the captured target nucleic acid that is used in a subsequentamplification process.

FIG. 3 illustrates a TSU primer complex, such as shown in FIG. 2 (3.),in more detail. The target strand is in a capture complex made up of thetarget strand, a capture probe that contains a 5′ target specificsequence (TS3) that hybridizes specifically to a complementary targetsequence (TS3′) and a 3′ poly-A sequence, shown hybridized to animmobilized probe that is a complementary poly-T sequence which isattached to a support. Vertical lines (∥∥|) are used to indicatehybridization between some of the complementary sequence regions. Thetarget strand is also attached to a TSU primer complex by hybridizationbetween the target's TS1′ sequence region and the complementary targetspecific sequence region (TS1) of the TSU promoter primer in the TSUprimer complex. The TSU primer complex is made up of the TSUnon-promoter primer hybridized at its U2 sequence region to thecomplementary U2′ sequence region of the S-oligonucleotide, which has a3′ blocked end (⊖), and the 5′ region of the S-oligonucleotide ishybridized at its U1′ sequence region to the complementary U1 sequenceregion in the TSU promoter primer that includes a 5′ promoter sequenceregion (P) and a 3′ TS1 region. The target strand contains a targetspecific sequence region (TS2), which is identical to the targetspecific sequence region (TS2) of the TSU non-promoter primer. All ofthe target specific regions of the target strand (TS1′, TS2 and TS3′)are independent sequences in the target strand.

FIG. 4 illustrates a preferred embodiment of a TSU primer complex,similar to one illustrated in FIG. 3, in which the upper strand is a TSUnon-promoter primer made up of a 3′ TS2 region and a 5′ universalsequence region, U2(+), which is hybridized to a 3′ complementary U2′sequence region of the S-oligonucleotide, which has a 3′ blocked endmade up of a 3′ to 3′ C linkage. The S-oligonucleotide contains anabasic spacer that links the 3′ U2′ sequence region to the 5′ U1′sequence region which is the complement of the U1(−) sequence region inthe TSU promoter primer, to which it is hybridized. The TSU promoterprimer includes a 5′ promoter sequence (P) and a 3′ target specificsequence region (TS1) that flank an internal U1 region. Preferredembodiments of this type of S-oligonucleotide include as the spacer anabasic compound, e.g., (C9).sub.2 or (C9).sub.3 that is covalentlyjoined to the flanking U1′ and U2′ sequences.

Although FIG. 2 illustrates only three different TSU primer complexesand capture probes (labeled TSUa, TSUb, and TSUc for Targets a, b and c,respectively) and only one target nucleic acid (Target a), it will beappreciated that many different TSU complexes and captureoligonucleotides, each specific for its own respective target nucleicacid, may be included in a TCR. And a sample may include many differenttarget nucleic acids, all of which may be selectively removed from othersample components. Thus, by including additional TSU primer complexesand probes in a TCR, but using substantially the same steps illustratedin FIG. 2, one or more different targets with attached TSU primers andcapture oligonucleotides each bound specifically to their respectivetargets, may be separated from the mixture by using one or more supportsthat bind to one or more target-primer complexes selectively. Forexample, different size particles may be used as supports, each with adifferent immobilized probe that selectively binds a target specificcapture probe, so that each desired target present in a single samplemay be selectively removed by size separating the supports with theirattached captured target and TSU primer complexes. Although FIG. 2illustrates capture probes that include poly-A regions to hybridize toimmobilized poly-T sequences, those skilled in the art will appreciatethat members of any specific binding pair may be used to capture atarget nucleic acid to a support, and different binding pair members maybe used to selectively isolate different targets from a complex samplemixture. For example, referring to FIG. 2, the TSUa primer complexesspecific to Target a, could be separated from the mixture by using a TSacapture probe that contains a ligand for receptor a in which receptor ais associated with the support as the immobilized probe. And, forexample, Targets a, b, and c all contained in one sample could beassociated with their respective TSU primers and separated from othersample components by using different combinations of binding pairmembers (BPM) on the capture probes (BPMa1, BPMb1, and BPMc1,respectively) which bind to immobilized probes via a specific bindingpair partner (BPMa2, BPMb2, and BPMc2, respectively), to captureindividually the targets, either all to the same support or to supportsspecifically for one or more targets determined by the second bindingpair partner(s) associated with the support(s). For example, a captureprobe for Target a associated with BPMa1 of avidin selectively removesTarget a from the sample by using an immobilized probe having a BPMa2 ofbiotin attached to a first support, whereas in the same TCR, a captureprobe for Target b is associated with a BPMb1 of an Fab fragment whichselectively removed Target b by using an immobilized probe having aBPMa2 of the ligand for the Fab fragment attached to a second support,where the first and second supports are separable by standardmethodologies. Supports with attached complexes that include the desiredtarget nucleic acids may be separated from the other components in themixture, including other sample components, such as cell debris,organelles, proteins, lipids, carbohydrates, other nucleic acids, andfrom unbound primers and capture probes. Any of a variety of well-knownways may be used to separate supports with attached complexes from othercomponents in the mixture, e.g. by centrifugation, filtration, gravityseparation, magnetic separation of magnetized materials, aspiration, andthe like. Thus, following target capture, only TSU primers bound totheir respective targets are carried into the amplification phase of theassay because unbound oligonucleotides are separated from the targetsduring the target capture phase. Additional washing step(s) may beincluded in the target capture phase to wash supports with the attachedtargets and primer complexes, thus further purifying the captured targetnucleic acids with attached TSU primers form other sample components andunbound oligonucleotides before the amplification phase.

Next, amplification is initiated by using the TSU primers specific forthe intended target nucleic acids, i.e., primers carried into theamplification mixture with the captured complex that includes the targetnucleic acid strand linked by hybridization to its corresponding TSUprimer(s). In some preferred embodiments, the TSU primers carried intothe amplification phase are in a TSU primer complex made up of a TSUpromoter primer, S-oligonucleotide, and TSU non-promoter primer for theintended target (see FIG. 1 and FIG. 2). Other TSU primers specific forother analytes that were absent from the sample, and therefore notcaptured, are discarded in the target capture stage and aresubstantially absent from the amplification reaction mixture. Thus, theinitial synthetic step in amplification relies on TSU primers attachedspecifically to the intended target nucleic acids present in at initialamplification phase. Because the TSU primers are already linkedspecifically to their intended target nucleic acid sequences,amplification initiates efficiently when other reaction components(e.g., enzymes and co-factors, synthetic substrates) are mixed with thecaptured target and its attached TSU primer or primer complex. The 3′end of the TSU promoter primer is extended synthetically as illustratedin FIG. 5 which shows the product that results from a first syntheticstep in the initial amplification phase, in which the 3′ end of the TSUpromoter primer, hybridized at its TS1 sequence to the TS1′ sequence ofthe target strand, has been synthetically extended to make a firststrand cDNA. For simplicity, the other components of a TSU primercomplex (the S-oligonucleotide and TSU non-promoter primer) have notbeen illustrated in FIG. 5, but it will be understood that the entireTSU primer complex may be attached to the RNA template strand duringthis synthetic step. Synthesis that initiates from the TSU promoterprimer on the RNA template strand uses an RNA directed DNA polymerase ofa reverse transcriptase (RT) enzyme supplied in the amplificationreaction mixture to synthesize a complementary DNA (cDNA) strand. Apreferred RT is one that includes RNAse H activity to degrade an RNAtarget/template strand, although the RNA dependent DNA polymeraseactivity and the RNA degradation activity may be supplied by differentenzymes in the amplification reaction mixture. The synthesized cDNAstrand contains a sequence TS2′ which is complementary to the TS2sequence in the target/template strand. Following synthesis of the cDNA,degradation of the RNA template strand occurs from the RNAse H activityin the reaction mixture, resulting in a single strand DNA that containsa 5′ promoter sequence, the U1 sequence and the TS1 sequence, allsupplied by the TSU promoter primer, and a 3′ sequence that containssequence complementary to the RNA template strand, including the TS2′sequence which is 3′ of the TS1, U1 and P sequences. This resulting cDNAstrand is shown in FIG. 6.

The first strand cDNA then binds to the TSU non-promoter primer byhybridization between the TS2′ sequence of the cDNA and thecomplementary TS2 sequence of the TSU non-promoter primer, which wascarried into the amplification reaction mixture as part of the TSUprimer complex bound to the captured target nucleic acid. In preferredembodiments, the isothermal amplification conditions maintain the TSUnon-promoter primer in a TSU primer complex (i.e., linked via theS-oligonucleotide to the TSU promoter primer) during the initial cDNAsynthesis step and then the 3′ TS2 portion of the TSU non-promoterprimer in the complex hybridizes to the cDNA strand. Such embodimentsare advantageous because they make use of efficient kinetics ofhybridization that performs substantially as intramolecularhybridization because the TS2 and TS2′ sequences are in close proximitydue to the maintained structure of the TSU primer complex joined to thecDNA. Referring to FIG. 7, the 3′ end of the TSU non-promoter primerhybridized the cDNA strand via hybridization of the TS2 and TS2′sequences is enzymatically extended by a DNA polymerase using the cDNAas a template strand to synthesize a second strand of DNA. Forsimplicity, FIG. 7 shows the TSU non-promoter primer without the othercomponents of the TSU primer complex as described above, but thosecomponents may be maintained during synthesis of the second strand DNA.The second strand DNA includes a 5′ universal sequence (U2) and TS2sequence, both contributed by the TSU non-promoter primer, a DNA strandextended from the 3′ end of the TSU primer, which includes a TS1′sequence and universal sequence U1′ (both complementary to the TS1 andU1 sequences, respectively, of the cDNA and the TSU promoter primer),and a 3′ sequence complementary to the promoter sequence (P) of the TSUpromoter primer. The resulting structure is a substantially dsDNA thatcontains a functional promoter sequence for its respective RNApolymerase enzyme.

Continuing the initial phase of isothermal amplification, as shown inFIG. 8, the RNA polymerase (RNA Pol) specific for the promoter sequencebinds to the functional promoter and initiates transcription from thesubstantially dsDNA, to make multiple RNA transcripts. These transcriptsinclude a 5′ U1 sequence, followed by the TS1 sequence, additionaltarget-specific sequence located between the TS1 and TS2′ sequences, theTS2′ sequence, and a 3′ U2′ sequence. The RNA transcripts contain targetspecific sequences flanked by a first universal sequence (U1), and asecond universal sequence (U2′), which differ from each other (one suchtranscript is illustrated in FIG. 9).

In the second phase of amplification, universal primers (UP1 and UP2 ofFIG. 1) are used to make additional RNA transcripts in a continuouscycle of isothermal amplification, using RNA transcripts as templatesfor synthesis of additional amplification products or amplicons.Preferred embodiments use the universal primers in an isothermalamplification reaction similar to TMA or NASBA reactions. In a firststep of the second phase of amplification, a universal non-promoterprimer (UP2), which consists essentially of a U2 sequence complementaryto the 3′ U2′ sequence of the RNA transcripts produced in the firstphase of amplification, hybridizes to the initial RNA transcripts (seeFIG. 9). The 3′ end of the UP2 primer is extended synthetically in anenzymatic isothermal reaction as illustrated in FIG. 10, in which theRNA transcripts from the initial phase of amplification enter the secondphase at the lower left. The RT enzyme binds and initiates cDNAsynthesis from the 3′ end of the UP2 primer by using the RNA directedDNA polymerase activity and the transcript as a template. Following thedark arrows in FIG. 10 illustrates the steps in the second phase ofamplification. The RNA template strand in the duplex with the cDNA isdegraded by RNAse H activity, allowing the cDNA to hybridize at the U1′sequence to the complementary U1 sequence of the universal promoterprimer (UP1). The RT binds to the 3′ end of the UP1 primer and initiatessecond strand DNA synthesis by using the DNA directed DNA polymeraseactivity and the cDNA strand as a template strand. The resulting dsDNAcontains a functional promoter sequence and, on each strand, twouniversal sequences flanking the target specific sequences. RNApolymerase (RNA Pol) specific for the promoter sequence binds to thefunctional promoter and makes 100 to 1000 transcripts (RNA amplicons)that are identical structurally to the initial RNA transcripts made inthe first phase of amplification. The additional transcripts serve astemplates for more iterations of the process. The RNA transcripts madein the second phase of amplification become available for use in theamplification process when they are made, i.e., no denaturation step isrequired, thus efficiently amplifying the universal and target specificsequences in a continuous isothermal process. RNA transcripts madeduring the second phase of the isothermal amplification process may bedetected during the reaction (i.e., in real time) or at a designated endpoint of the reaction (e.g., a specific time after beginning theamplification reaction or when amplification substantially terminatesdue to exhaustion of substrates present in the reaction).

The RNA amplicons may be detected by using well-known detection methodswhich may detect simply an increase in nucleic acid concentration or maydetect selected amplified sequences. For example, detection mayspecifically detect one or more of the universal sequence(s) orsubsequence(s) thereof, or a target specific sequence(s) or asubsequence thereof, or a contiguous sequence that combines portions ofuniversal and target specific sequences. Preferably, a detection stepthat uses a probe for detection of amplicons allows homogeneousdetection, i.e., detection of the hybridized probe without removal ofunhybridized probe from the mixture (e.g., U.S. Pat. Nos. 5,639,604 and5,283,174,). In preferred embodiments that detect the amplified productnear or at the end of the second phase of amplification, a linear probeis used to provide a detectable signal that indicates hybridization ofthe probe to the amplified product. In preferred embodiments that detectthe amplified product in real time, the probe is preferably a probe inwhich signal production is linked to the presence of the targetsequence, such as a molecular beacon, molecular torch, or hybridizationswitch probe, that is labeled with a reporter moiety that is detectedwhen the probe binds to amplified product. Such a probe may include alabel, e.g., a fluorophore attached to one end of the probe and aninteracting compound, e.g., a quencher attached to another location ofthe probe to inhibit signal production from the label when the probe isin a “closed” conformation that indicates it is not hybridized to theamplified product, whereas detectable signal is produced when the probeis in “open” conformation that indicates it is hybridized to theamplified product. Various probe structures and methods of using themhave been described previously (e.g., U.S. Pat. Nos. 5,118,801,5,312,728, 5,925,517, 6,150,097, 6,849,412, 6,835,542, 6,534,274, and6,361,945, US App. 2006-0068417 and PCT App WO 2006/093892).

The methods of target capture and amplification that use at least oneuniversal sequence described herein may be performed in a variety ofdifferent ways. In some preferred embodiments, all of the steps areperformed substantially in a liquid phase, i.e., one in which most orall of the steps occur with the components in the reactions beingpresent in substantially aqueous media. For example, the steps of targetcapture may be performed in a substantially liquid aqueous mixture thatallows hybridization of the capture probe to the target nucleic acid andthe capture probe to an immobilized probe in solution phase by usingimmobilize probes attached to small particles or beads that are mixed orsuspended in the solution phase. Similarly, in some preferredembodiments, all of the amplification steps are performed by having allof the amplification components (e.g., substrates, templates, enzymesand cofactors) in a solution phase for the entire reaction. Thedetection step that detects a signal resulting from the presence ofamplified products may also be performed in a substantially aqueoussolution phase (e.g., as described in U.S. Pat. Nos. 5,639,604 and5,283,174). In other preferred embodiments, one or more of the steps inan assay that includes target capture, amplification and detection stepsmay be performed substantially attached to a solid phase, such as asupport matrix or particle, to compartmentalize or localize detection ofa particular analyte of interest. Such embodiments are advantageousbecause amplification products may be localized, e.g., temporally orspatially, for separate detection of signals resulting from the presenceof one or more selected analytes present in a sample. This isparticularly useful when a sample may contain multiple differentanalytes that are all treated in substantially the same reagent mixturesduring target capture, amplification and/or detection steps, but forwhich separate detection of signals resulting from the presence ofamplified products for each analyte is desired.

Referring to FIG. 11, two preferred embodiments are illustrated thatallow assay steps to be performed attached to a support. Bothembodiments use a combination of TSU primers (TSU promoter primer andTSU non-promoter primer sequences) that are attached via members of aspecific binding pair to a support. The TSU primers in both embodimentsprovide target specific sequences (TS1 and TS2) and universal sequences(U1 and U2) as described earlier in this disclosure. And bothembodiments use universal primers (UP1 and UP2) in the second phase ofamplification as described earlier in this disclosure. In contrast tothe embodiments that use a TSU primer complex that includes anS-oligonucleotide (e.g., as shown in FIG. 3), the TSU primers of thesetwo embodiments are physically linked by being attached to a support. InFIG. 11, embodiment 1, the TSU promoter primer and TSU non-promoterprimer sequences are linked to a support via a first binding pair member(BPM1) that binds specifically with a second binding pair member (BPM2)attached to the support. This may be accomplished by synthesizing asingle oligonucleotide that contains all of the structural elements ofthe TSU promoter primer and TSU non-promoter primer sequences in theappropriate order (e.g., 3′-T52-U2-5′-5′-P-U1-TS1-3′) with a BPM1element associated with the synthetic oligonucleotide, or bysynthesizing two oligonucleotides (TSU promoter primer sequence and TSUnon-promoter primer sequence) which are then attached to the same BPM2moiety via a BPM1 moiety associated with the primers. In FIG. 11,Embodiment 2, the TSU promoter primer oligonucleotide and TSUnon-promoter primer oligonucleotide are linked to the same support via afirst binding pair member (BPM1) associated with each primer that bindsspecifically but independently with a second binding pair member (BPM2)attached to the support. In both embodiments, the TSU primers aremaintained in close proximity by being bound to the same support.Because the TS1 sequence of the TSU promoter primer binds with acomplementary sequence in the target nucleic acid strand (TS1′), the TSUprimer may function as a capture probe to selectively bind and separatethe intended target nucleic acid from a sample mixture, by using thesupport to separate the TSU primer-target complex from other samplecomponents. Then, the TSU primer-target complex attached to the supportand mixed with amplification reaction components (e.g., substrates,enzymes, cofactors) serves as a primer-template complex in the initialphase of amplification substantially as described earlier in thisdisclosure except that the support substitutes for the S-oligonucleotidein providing the TSU non-promoter primer in close proximity to the cDNAsynthesized from the initial TSU primer-target complex. The RNAtranscripts from the first phase of amplification then serve astemplates for the second phase of amplification by using the UP1 and UP2universal primers substantially as described in this disclosure(referring to FIG. 10).

The supports in both embodiments shown in FIG. 11 may be used tolocalize the amplification and detection steps, temporally or spatiallyor both for specific analytes of interest. For example, if threedifferent analytes (A1, A2, A3) are present in a sample, the threedifferent target nucleic acids (T-A1, T-A2, T-A3) may be captured in asingle target capture step by using three different TSU primers attachedto different supports or different locations of one support, each TSUprimer specific for its respective analyte by use of different TS1sequences (TS-A1, TS-A2, TS-A3), each specific for one of the targets.Spatial separation of may result, e.g., when a single support is used towhich the TSU primer complexes are attached at different predeterminedloci, such as in an array. Other embodiments that achieve spatialseparation include different wells or containers of a multi-chambereddevice which contain TSU primer complexes in a predetermined pattern ora random pattern, such as achieved by dispensing a known amount ofsolution in which one or more support particles are suspended at apredetermined probability, e.g., a dilution at which an average of oneor fewer individual supports are deposited at a locus on or in a well orchamber. Spatial separation may also be achieved by selectivelyseparating each of the supports into separate chambers or sections of adevice before performing the amplification step by using a physicalcharacteristic of the support to which each of the different TSU primersis attached. For example, TSU primers having different TS1 sequences(TS-A1, TS-A2, TS-A3) may be attached to different particular supportsthat are separable based on size, density, ligand binding capabilities,magnetic properties and the like, so that the different supports withtheir attached TSU primer-target complexes may be spatially separatedbefore performing amplification steps that all use the same reagents,including the same universal primers. The amplified product detected ata particular spatial location in the detecting step indicates whether aparticular analyte was present in the sample, and the cumulativedetection results of all of the locations may indicate that more thanone analyte was present in the sample, and may provide a quantitative orproportional measurement of each analyte present in the sample. Forexample, if an array of 100 chambers is used in which three differentTSU primer-target complexes (i.e., TS-A1, TS-A2, TS-A3 primers) arespatially separated to produce an average of one TSU primer-targetcomplex per locus before performing amplification steps, and thedetection step results in 10 chambers positive for the TS-A1 primer, 30chambers positive for the TS-A2 primer, and 50 chambers positive for theTS-A3 primer, then the results indicate that the sample contained allthree analytes A1, A2 and A3, in a ratio for A1:A2:A3 of 1:3:5.

Similarly, temporal separation may be used to amplify products fromdifferent target nucleic acids and detect the amplified products. Foreither embodiment of FIG. 11, using the model system of three differentanalytes (A1, A2, A3) present in a sample, the three different targetnucleic acids (T-A1, T-A2, T-A3) may be captured in a single targetcapture step by using three different TSU primer complexes attached tosupports, each TSU primer complex specific for its respective analyte byuse of different TS1 sequences (TS-A1, TS-A2, TS-A3). Amplification inthe first and second phases is performed substantially as describedpreviously herein, except that at different times during theamplification a detection measurement is made for each of the amplifiedproducts, e.g., at a first time (T1) for the A1 product, at a secondtime (T2) for the A2 product, and at a third time (T3) for the A3product, which each product results in a different detectable signalsuch as fluorescence at a different wavelength. Thus, positive signalsdetected only at T1 and T3 indicate that the sample contained onlyanalytes A1 and A3, and did not contain A2. In other embodiments,temporal detections may be made at sequential times over an extendedtime range during the amplification reaction, e.g., at T1, T4 and T7 forA1, at T2, T5 and T8 for A2, and at T3, T6 and T9 for A3, and thecumulative results may indicate both the presence and relative amountsof each of the analytes present in a sample. For example if a positivesignal is detected at T1, T4 and T7 it indicates for A1 is present inthe sample, and a positive signal is detected at T8 it indicates that A2is present in the sample, and a positive signal is detected at T6 and T9it indicates that A3 is present in the sample. Amplification for each ofthe analytes is expected to proceed at approximately the same rate dueto use of the same conditions and universal primers in the second phaseof amplification. Thus the relative amount of amplified product and theresulting earliest time of signal detection for each amplified productprovides an indication of the proportional amount of each of theanalytes present in the sample. Based on the model system results abovein which signal for A1 is detected before signal for A3, which isdetected before signal for A2, the relative of amounts of each of theanalytes in the sample are A1 greater than A3 greater than A2.

A combination of spatial and temporal separations may be used in anassay to amplify and selectively detect amplified products from morethan one analyte in a reaction, to allow detection of amplified productsfor an analyte at discrete locations and times. For example, spatialseparation may involve use of an array of TSU primer complexes attachedto a support at predetermined loci combined with temporal separation bydetecting signals at different time points from each or selected groupsof loci to detect amplification products resulting from an amplificationreaction performed on the array. In another embodiment, TSU primercomplexes attached to particulate supports may be suspended in solutionphase of an amplification reaction mixture for some portions of theamplification reaction and then sedimented or attracted to a surface ina random or non-random pattern (spatial separation) for detection ofsignal from the localized amplification products made during otherselected times during the amplification reaction (temporal separation)so that the resulting series of cumulative patterns of detectablesignals provide information on both the presence and relative amounts ofanalyte(s) present in the sample. Those skilled in the art willappreciate that a wide variety of spatial, temporal, and combinedspatial and temporal separations may be used to selectively detectamplification products resulting from amplification reactions thatinclude multiple analytes (i.e., multiplex reactions).

Those skilled in the art will also appreciate that other embodiments areencompassed by the general principles of the assays disclosed herein.That is, assays that include a target capture step to separate a targetnucleic acid from a sample and attach an initial TSU primer to theselected target nucleic acid, followed by an isothermal amplificationreaction that is characterized by two phases, in which the first phaseintroduces universal sequences into products made from the targetnucleic acid, and the second phase uses those universal sequences forfurther production of amplification products, which are detected in thefinal stage of the assay. The target capture step includes attachment ofan initial TSU primer that contains a first universal sequence attachesto the target nucleic acid. The target capture step is followed by aninitial phase of isothermal amplification that uses the initial TSUprimer and a second TSU primer, which contains a second universalsequence, to produce RNA transcripts that contain the first universalsequence and the complementary sequence of the second universalsequence, which flank a target specific sequence. This is followed by asecond phase of isothermal amplification in which the RNA transcriptsmade in the first phase are amplified by using a continuous process ofmaking additional RNA transcripts by using universal primers that bindspecifically to the universal sequences (or their complements)introduced by using the initial TSU and second TSU primers. The finaldetection step detects a signal resulting from the amplified productsmade during the second phase of isothermal amplification to indicatethat the target nucleic acid selected in the target capture step waspresent in the tested sample. These general assay steps may be used witha variety of different primers of different sequences which can bereadily designed by those skilled in the art of molecular biology inview of the general structural features of the primers described herein.

Other embodiments of isothermal amplification methods that use universalsequences may use fewer TSU primers and universal primers compared tothe embodiments described above, while retaining features characteristicof the method such as attachment of a TSU primer to the target nucleicacid during the target capture step and but performing isothermalamplification steps by using a combination of universal and targetspecific primers. For example, an embodiment may using only one initialTSU promoter primer which hybridizes to the target nucleic acid duringthe target capture step and is extended synthetically to introduce asingle universal sequence into the cDNA and later into the RNAtranscripts made during the first phase of isothermal amplification, sothat the second phase of amplification uses only a single universalprimer combined with one or more target specific primers to make theamplification products that are detected to indicate the presence of theanalyte(s) in the tested sample. FIG. 12 illustrates two embodiments(Embodiment 1, upper, and Embodiment 2, lower) to compare difference inthe (A.) target capture (TC) step with initial primer attachment and(B.) primers used in the second phase of amplification. Referring toFIG. 12, Embodiment 1 in the TC step attaches to the target strand a TSUprimer complex that includes both a TSU promoter primer and a TSUnon-promoter primer linked by an S-oligonucleotide as described earlierherein, where the target specific portion of the TSU promoter primerbinds to a complementary sequence in the target strand to link auniversal sequence (U1) to the cDNA that will be made by extending the3′ end of the TSU promoter primer in the first phase of isothermalamplification, as described earlier herein. In contrast, Embodiment 2 inthe TC step attaches to the target strand only a TSU promoter primerwhich is hybridized to via its target specific portion to acomplementary sequence in the target strand to link a U1 sequence to thecDNA that will be made by extending the 3′ end of the TSU promoterprimer, as described above. In Embodiment 1, the first phase ofamplification will continue as described earlier with reference to FIGS.5 to 8, in which the TSU non-promoter primer with its universal sequencewill be used to make the second DNA strand, so that the RNA transcriptsmade in the first phase of amplification will contain two universalsequences. In Embodiment 2, instead of using a TSU non-promoter primer,a target specific non-promoter primer is hybridized to a complementarysequence in the cDNA and extended synthetically to make the secondstrand DNA, so that the RNA transcripts made in the first phase ofamplification contain only one universal sequence. Referring to FIG. 12,B., in the second phase of isothermal amplification for Embodiment 1(upper portion), two universal primers, a universal promoter primer(UP1) and universal non-promoter primer (UP2), are used to make RNAamplicons as described earlier with reference to FIG. 10. In contrast,in Embodiment 2, of FIG. 12, B., the second phase of isothermalamplification uses only one universal promoter primer (UP1) combinedwith a target specific primer (TSP). Referring to FIG. 13, in the secondphase of isothermal amplification, RNA amplicons are made by usingsynthetic steps similar to those described above, but by using the TSP(instead of UP2) to initiate synthesis of the cDNA using the RNAtranscripts as templates (starting at lower left in FIG. 13.). That is,in this embodiment, no U2 or U2′ universal sequences are present in thereaction.

An embodiment that uses a single TSU primer and a target specific primermay be used in assays that make use of the TSU primer attached to asupport, similar to those embodiments described above with reference toFIG. 11. FIG. 14 schematically depicts a TSU promoter primeroligonucleotide made up of a promoter sequence (P), a universal sequence(U1) and a target specific sequence (TS1) which is attached to a supportvia a first binding pair member (BPM1) which binds specifically to asecond binding pair member (BPM2) attached to the support. The TSUpromoter primer is used in the first phase of amplificationsubstantially as described above with reference to FIG. 12 (Embodiment2). For the second phase of amplification, a mixture containing auniversal promoter primer (UP1) and a target specific primer (TSP) isused, as shown in FIG. 14, using the steps as described above anddiagramed in FIG. 13, to amplify the RNA transcripts. In one preferredembodiment, a TSU promoter primer attached to a support (as in FIG. 14)may be used to capture the target nucleic acid strand to which ithybridizes by using its TS1 sequence that is complementary to a sequence(TS1′) in the target strand. Alternatively, an embodiment that uses asingle TSU primer attached to a support may be used in combination witha TC step that uses a capture complex (as in FIG. 12, A.) that includesa support, an immobilized probe and a target specific capture probe, asdescribed in detail previously. In an embodiment that uses a TSUpromoter primer attached to a support as the means for separating thetarget nucleic acid from other sample components, then the TSU promoterprimer serves essentially as the capture probe and as the primer forinitiation of cDNA synthesis when the complex that includes the supportand the TSU promoter primer hybridized to the target strand is mixedwith other amplification reagents. In an embodiment that performs a TCstep that uses a capture complex made up of a capture probe hybridizedto the target strand and bound to the immobilized probe attached to thesupport, then the TSU promoter primer hybridized to the target strandand attached to another support acts as the primer for initiation ofcDNA synthesis when the complex is mixed with other amplificationreagents. In both embodiments, the TSU primer attached to a support maybe used to separate amplification products spatially, temporally, or asa combination of spatial and temporal separation as described above withreference to FIG. 11, except that the second phase of isothermalamplification relies on using a TSP instead of a universal primer (UP2).

Embodiments such as those described with reference to FIGS. 12(embodiment 2), 13 and 14, that use a TSU promoter primer in combinationwith a target specific primer (TSP) are advantageous in a number ofapplications. For example, in assays for detection of one or morespecies or isolates that share a common target sequence (TS1′) that isconserved among the different targets, a TSP may be included for each ofthe different targets by making the TSP sequence specific for eachtarget. For example, a TS1′ sequence that occurs in 16S or 23S rRNAsequence of many members of a genus (e.g., Mycobacterium) may be used todesign a TSU promoter primer that contains a TS1 sequence that will bindto the target 16S or 23S rRNA from all of the intended targets in thegenus. Then, for each of the intended target species included in thegenus targets (e.g., M. tuberculosis, M. avium, M. abscessus, M.africanum, M. asiaticum, M. avium, M. bovis, M. celatum, M. chelonae, M.flavescens, M. fortuitum, M. gastri, M. gordonae, M. haemophilum, M.intracellulare, M. interjectum, M. intermedium, M. kansasii, M.malmoense, M. marinum, M. non-chromogenicum, M. paratuberculosis, M.phlei, M. scrofulaceum, M. shimodei, M. simiae, M. smegmatis, M.szulgai, M. terrae, M. triviale, M. tuberculosis, M. ulcerans or M.xenopi) a TSP specific for each member is designed and used in theisothermal amplification reaction to make amplified products specificfor each target species, which may be individually detected by usingstandard probe hybridization or size separation methods. In anotherexample, related viral targets, such different human papillomavirus(HPV) types may be detected in a single reaction mixture designing a TSUpromoter primer that binds via its TS1 sequence to a common sequence(TS1′) present in all of the desired HPV types to be detected (e.g., HPVtypes 16, 18, 31, 33, 35, 45, 51, 56, 58, 59 and 68). Thus, the initialcDNA made from the TSU promoter primer will be synthesized for each ofthe intended target HPV types present in the sample using HPV mRNA inthe E6/E7 gene target sequence. Then, for amplification and detection ofindividual HPV types of interest, a TSP is designed for each target(e.g., one each for HPV16 and HPV18) or for a combination of relatedtargets (e.g. one specific for both HPV 16 and HPV18), i.e., each TSPbinds specifically to a sequence of its intended HPV type(s) only. EachTSP specific for its target type is used in the isothermal amplificationreaction to make amplified products specific for the selected targettypes and the amplified products are individually detected by usingstandard methods (hybridization, size separation, sequencing) toidentify the HPV type(s) present in the tested sample. Embodiments suchas these are particularly useful for multiplex reactions, in which morethan one selected target is present in a sample and is amplified toproduce a detectable amplified product that is distinguishable fromother amplified products, so that a signal from each amplified productpresent in the reaction mixture indicates the target analytes that werepresent in the tested sample.

Another application for which embodiments that use a single universalsequence provided by a TSU primer combined with multiple target specificprimers (TSP) are useful is for detecting different forms of relatedgenetic sequences or products. For example, cancers may be correlatedwith the presence of certain genetic translocations or translocationbreakpoints (e.g., chronic myelogenous leukemia (CML) associated withtranslocations between human chromosomes 9 and 22 in the abl gene ofchromosome 9 and the “breakpoint cluster region” or bcr gene ofchromosome 22). To detect different types of translocations, anembodiment of the methods described herein uses a TSU primer in whichthe TS1 sequence is specific for a target sequence in a genetic sequenceor mRNA of one of the translocation members (e.g., abl gene) that iscommon to many different cancer-associated translocations, and thereforecan amplify sequences from many different translocations independent ofthe breakpoint. To amplify and detect specific translocations that areassociated with cancers or have particular prognostic value, a varietyof different TSPs are designed (e.g., different bcr sequences), each onespecific for amplifying a particular sequence associated with acancer-associated translocation, where the amplified sequence may bedetected specifically using standard methods (e.g., probe hybridization,sequencing, or size of amplicon). A sample suspected of containingnucleic acid (DNA or RNA) that has a diagnostic translocation sequenceis then amplified using the TSU promoter primer that amplifies manytranslocations in the target and with the many different TSPs,preferably in a single or a few multiplex reactions, and the amplifiedproducts are detected specifically to provide diagnostic or prognosticinformation based on the particular translocation sequences that areamplified and detected.

Similarly, embodiments that use a single universal sequence provided bya TSU primer and multiple target specific primers (TPS) are useful fordetecting different forms of related genetic sequences that occur indifferent expression products of a gene (e.g., PCA3 gene associated withprostate cancer; see U.S. Pat. No. 7,008,765). Such different expressionproducts may result from different splicing events in RNA transcripts,where some spiced RNAs are diagnostic of a disease or provide prognosticvalue, such as whether a cancer tissue is benign or malignant. In suchembodiments, a TSU promoter primer is designed to contain a TS1 sequencethat is specific for a TS1′ sequence contained in all or many forms ofthe differentially spliced RNA, and the multiple TSPs are designed toeach amplify only one form of the differentially spliced RNAs. Followingamplification using the TSU promoter primer and the TSPs, preferably ina single multiplex reaction mixture, the amplified products are detectedin a way that distinguishes them to provide information on theparticular form(s) of spliced RNA present in the tested sample.

Other embodiments that use a single universal sequence provided by a TSUprimer and multiple target specific primers (TPS) are useful fordetecting mutations in genetic sequences that provide diagnostic orprognostic information, such as by detecting the presence of one or moresequences that result in drug resistance. For example, a number of HIV-1mutations are associated with the viral infection being resistant totreatment with particular drugs (e.g., see U.S. Pat. No. 6,582,920, Yanget al.). To detect one or more drug resistance mutations in a singlereaction, the TSU primer is designed to contain a TS1 sequence that iscomplementary to HIV-1 mRNA that is common to HIV-1 strains andisolates, independent of whether the strain or isolate contains a drugresistance mutation. The multiple TSPs are designed to amplify aparticular sequence that contains a mutation associated with drugresistance. In some embodiments the TSPs are specific for the drugresistance mutations themselves, whereas in other embodiments, the TSPsare specific for a sequence that does not contain the drug resistancemutation per se, but which amplifies a product that contains the drugresistance mutation. The TSU promoter primer is used with the multipleTSPs, preferably in a single multiplex reaction, to amplify productsthat provide information on whether a drug resistance mutation waspresent in the nucleic acid of the tested sample. For example, forembodiments in which the TSPs are specific for each of the drugresistance mutations to be detected, the presence or absence of thedistinguishable amplified products indicates which mutations are presentin the tested sample. In other embodiments in which the TSPs arespecific for a sequence that does not contain the drug resistancemutation per se, but which amplifies a product that contains the drugresistance mutation(s), then standard methods of detecting themutation(s) are used, e.g., probe hybridization including on an array,sequencing, or size separation, including mass spectrometry.

Testing of embodiments that use TSU primers, TSU primer complexes anduniversal primers, in the isothermal amplification methods as describedherein has been performed and amplified products have been successfullydetected for viral targets and genetic sequences associated with cancermarkers, such as prostate specific antigen (PSA; e.g., U.S. Pat. No.6,551,778) and PCA3 sequences.

Those skilled in the art of molecular biology will appreciate that TSUoligonucleotides as described herein do not require any specificsequences to function, so long as the chosen sequences fulfill thefunctional requirements of the TSU oligonucleotides. That is, no singlesequence is required for any functional portion of a TSUoligonucleotide, e.g., no particular primer is required for a TSUpromoter primer or promoter provider, so long as the TSU oligonucleotidecontains sequences for all of the functional portions needed for itsfunction for the embodiment for which it is intended as disclosedherein. Similarly, a TSU primer that does not contain a promotersequence does not require any particular sequence so long as it containsa U sequence and a TS sequence that allows it to function for theembodiment for which it is intended as disclosed herein. Similarly, noparticular sequence is required for an S-oligonucleotide, a covalentlylinked oligonucleotide made up of two TSU oligonucleotide sequences, orfor two TSU oligonucleotides that are directly hybridized to each othervia complementary sequences, so long as the appropriate sequences foreach functional portion are included as described for the embodimentsdisclosed herein. Universal primers similarly do not require aparticular sequence but instead are chosen to contain sequences thatperform with the U sequence(s) chosen for the TSU oligonucleotides asdescribed herein. For example, a universal promoter primer or promoterprovider oligonucleotide contains a promoter sequence and a U sequencethat functions in the methods described herein, where the U sequence ofthe universal primer and the U sequence of the TSU promoteroligonucleotide are substantially identical. A U sequence in theuniversal primer may vary from the U sequence of the TSUoligonucleotide, so long at these sequences share enough identity toallow specific hybridization of the universal primer to a universalsequence once incorporated into an initial or subsequent amplificationproduct, forperforming in the methods disclosed herein. Similarly, theuniversal primer does not rely on any particular sequence but isselected to be substantially identical to the universal sequence of theTSU non-promoter primer with which it is used. Promoter sequences arepreferably, but not necessarily, the same in all TSU promoter primers orpromoter providers used in an assay for multiple targets because thatsimplifies other reaction components (i.e., a single RNA polymerase isused), but different promoter sequences that function with the same ordifferent RNA polymerases may be used. Those skilled in the art willappreciate that many different sequences may be incorporated into TSUoligonucleotides, S-oligonucleotides, and universal primers that fallwithin the scope of the compositions described herein, which thoseskilled in the art of nucleic acid amplification are capable ofselecting based on the descriptions of the structural and functionalfeatures of the oligonucleotides as described herein, wherefunctionality may be demonstrated by using routine testing methods.

Embodiments of the compositions and methods described herein may befurther understood by the examples that follow. Method steps used in theexamples have been described herein and the following informationdescribes typical reagents and conditions used in the methods with moreparticularity. Those skilled in the art of nucleic acid amplificationwill appreciate that other reagents and conditions may be used that willnot substantially affecting the process or results so long as guidanceprovided in the description above is followed. For example, althoughtranscription mediated amplification (TMA) methods are described thatuse a promoter primer or promoter provider oligonucleotide and anon-promoter primer in an initial phase of amplification, other methodsof transcription associated nucleic acid amplification in vitro thatrely on primer extension could be modified to use the TSUoligonucleotides as described herein to make amplified products by usinguniversal primers, i.e., the methods are not limited to TMA-basedembodiments. Those skilled in the art of molecular biology will alsounderstand that the disclosed methods and compositions may be performedmanually or in a system that performs one or more steps (e.g.,pipetting, mixing, incubation, and the like) in an automated device orused in any type of known device (e.g., test tubes, multi-tube unitdevices, multi-well devices such as 96-well microtitre plates, and thelike).

Exemplary reagents used in the methods described in the examples includethe following. Sample Transport Medium (“STM”) contained 15 mM sodiumphosphate monobasic, 15 mM sodium phosphate dibasic, 1 mM EDTA, 1 mMEGTA, and 3% (w/v) lithium lauryl sulfate (LLS), at pH 6.7. SpecimenDilution Buffer contained 300 mM HEPES, 3% (w/v) LLS, 44 mM LiC1, 120 mMLiOH, 40 mM EDTA, at pH 7.4. Target Capture Reagent (TCR) contained 250mM HEPES, 310 mM lithium hydroxide, 1.88 M lithium chloride, 100 mMEDTA, at pH 6.4, and 250.micro.g/ml of magnetic particles (1 micronSERA-MAG.SUP.™ MG-CM particles, Seradyn, Inc. Indianapolis, Ind.) with(dT).sub.14 oligomers covalently bound thereto. TC Wash Solutioncontained 10 mM HEPES, 150 mM sodium chloride, 6.5 mM sodium hydroxide,1 mM EDTA, 0.3% (v/v) ethanol, 0.02% (w/v) methyl paraben, 0.01% (w/v)propyl paraben, and 0.1% (w/v) sodium lauryl sulfate, at pH 7.5. ProbeReagent contained one or more labeled detection probes in a solutionmade up of either (1) 100 mM lithium succinate, 3% (w/v) LLS, 10 mMmercaptoethanesulfonate, and 3% (w/v) polyvinylpyrrolidon, or (2) 100 mMlithium succinate, 0.1% (w/v) LLS, and 10 mM mercaptoethanesulfonate.Hybridization Reagent was either (1) 190 mM succinic acid, 17% (w/v)LLS, 100 mM lithium hydroxide, 3 mM EDTA, and 3 mM EGTA, at pH 5.1, or(2) 100 mM succinic acid, 2% (w/v) LLS, 100 mM lithium hydroxide, 15 mMaldrithiol-2, 1.2 M lithium chloride, 20 mM EDTA, and 3.0% (v/v)ethanol, at pH 4.7. Selection Reagent used to treat mixtures that useAE-labeled detection probes contained 600 mM boric acid, 182.5 mM sodiumhydroxide, 1% (v/v) octoxynol (TRITON.sup.® X-100), at pH 8.5, andDetection Reagents used to elicit a chemiluminsecent signal fromAE-labeled probes included (1) Detect Reagent I made of 1 mM nitric acidand 32 mM hydrogen peroxide, and (2) Detect Reagent II (to neutralizepH) which was 1.5 M NaOH. An exemplary Amplification reagent orpre-amplification reagent, as used herein, can include a mixture atabout pH=7.5 to 8.0 and containing about 25 to 27 mM Tris; about 17 to23 mM MgCl.sub.2; about 23 to 30 mM KCl; about 3 to 7.5% v/v glycerol;about 0.04 to 0.05 mM Zn Acetate; about 0.5 to 0.7 mM of each of dATP,dCTP, dGTP, dTTP; about 3.9 to 5.4 mM of each of rATP, rCTP, rGTP, rUTP;and about 0.015 to 0.02% v/v ProClin 300 preservative (Sigma Aldrich,St. Louis, Mo.). Primers and/or probes may be added to the reactionmixture in the amplification reagent or are separate from the reagent(primerless amplification reagent or primerless pre-amplificationreagent). Exemplary Enzyme reagents, as used in amplification orpre-amplification reaction mixtures, can include a mixture at aboutpH=7.0 and containing about 56 to 224 U/.micro.l of MMLV reversetranscriptase (RT); about 35 to 40 U/.micro.l of T7 RNA polymerase perreaction (where 1 U of RT incorporates 1 nmol of dTTP in 10 min at37.deg.C using 200-400 micromolar oligo dT-primed polyA template, and 1U of T7 RNA polymerase incorporates 1 nmol of ATP into RNA in 1 hr at37.deg.C using a T7 promoter in a DNA template); about 4 to 16 mM HEPES;about 17 to 70 mM N-Acetyl-L-Cysteine; about 0.75 to 3.0 mM EDTA; about0.01 to 0.05% w/v Sodium Azide; about 20 to 25 mM Trizma; about 30 to 50mM KCl; about 7.5 to 20% v/v glycerol anhydrous; about 2.5 to 10% v/vTriton-X 102 and 0 to about 150 mM trehalose.

An exemplary protocol for TMA reactions that detect results by usinglabeled probes at the end of the amplification reaction follows. The TMAreaction uses substantially the procedures described previously indetail (U.S. Pat. Nos. 5,399,491 and 5,554,516). Briefly, a reactionmixture (e.g., 0.08 ml) containing amplification reagent, target nucleicacid, and amplification oligomers (e.g., 15 pmol of each oligomer perreaction) was mixed, covered with silicon oil (0.2 ml) to preventevaporation, and incubated for 10 min at 62.deg.C and then for 5 min at42.deg.C, and then the enzyme reagent (0.025 ml containing reversetranscriptase and T7 RNA polymerase) was added, and reaction mixtureswere incubated for 60 min at 42.deg.C. Following amplification,detection of the amplified products involved mixing the amplificationmixture with an acridinium ester (AE) labeled detection probe oligomerspecific for the amplification product (e.g., 0.1 pmol per reaction in0.1 ml of probe reagent, or an amount previously determined to produce amaximum detectable signal in an acceptable range, such as up to2,000,000 relative light units (“RLU”) from hybridized labeled probe).Mixtures of probe and amplified sequences were incubated to bind theprobe to the amplified product and then treated to producechemiluminescent signal from hybridized probes substantially asdescribed (U.S. Pat. Nos. 5,283,174 and 5,639,604). Briefly, the probeand amplified product mixtures were incubated for 20 min at 62.deg.C,then cooled at room temperature about 5 min and selection reagent (0.25ml) was added, mixed, incubated 10 min at 62.deg.C and then at roomtemperature for 15 min to hydrolyze the AE label on unbound probes.Chemiluminescence from AE on bound probes was produced by adding detectreagent I, incubating, adding detect reagent II, and measuringchemiluminescence in a luminometer (e.g., LEADER.sup.®, Gen-Probe Inc.,San Diego, Calif.).

An exemplary protocol for TMA reactions that detect results in real timefollows. The assay includes purification of target nucleic acids beforeamplification, amplification, and detection of the amplified productsduring amplification. Target capture is performed substantially aspreviously described in detail (U.S. Pat. Nos. 6,110,678, 6,280,952, and6,534,273). Briefly, samples were prepared to contain known amounts oftarget RNA (in vitro transcripts (“IVT”) present at a predetermined copylevel per sample in a total volume of 0.2 ml of a 1:1 (v:v) mixture ofwater and sample transport medium). Each sample was mixed with 0.05 mlof TCR that typically contained 5 to 15 pmol of target capture oligomer(TCO) specific for the analyte nucleic acid to be captured (i.e., 3′target-specific binding region) and a 5′ tail region (e.g.,dT.sub.3A.sub.30 sequence) for binding to the immobilized probe (e.g.,poly-T oligomers attached to paramagnetic particles; 12.5.micro.g ofparticles with attached oligomers per reaction), 5 to 15 pmol of TSUprimer and/or complex that includes TSU primer and TSU promoter primeror provider sequence for each analyte (for initial phase ofamplification), and optionally 2 to 5 pmol of blocker oligomer (for rTMAamplification reactions). The mixtures were incubated for 25 to 30 minat 60±1.deg.C and then for 25 to 30 min at room temperature (20 to25.deg.C) to form hybridization complexes through which target nucleicacids were bound to the paramagnetic particles which were the isolatedby using magnetic separation (e.g., KingFisher96.sup.™ magnetic particleprocessor, Thermo Fisher Scientific, Inc., Waltham, Mass.) and washedone time using TC wash solution. Particles were resuspended in 0.06 to0.1 ml of amplification reagent and with amplification oligonucleotidesused in the second phase of amplification (e.g., TS primer, universalprimer(s), 3′ blocked universal promoter provider). Detection probes(e.g., molecular beacon or molecular torch probes labeled with afluorescent label compound) may be added with amplificationoligonucleotides, or with addition of enzymes, or following addition ofenzymes. Reaction mixtures were covered to prevent evaporation andincubated for 1 to 2 minutes at 42±0.5.deg.C. While keeping them at42±0.5.deg.C, the mixtures were uncovered and mixed with 0.02 ml ofenzyme reagent per mixture, covered again, and incubated for 30 to 90minutes at 42±0.5.deg.C, during which time fluorescence is measured atregular time intervals (e.g., every minute) which are referred to as“cycles” for data collection and display, which is typically a graph ofdetected fluorescence units versus time (cycles), from which a time ofemergence of signal was determined (i.e., time at which fluorescencesignal for a sample becomes positive over a background level, which isusually predetermined for the assay).

Example 1: Universal TMA (uTMA) System for Detection of Multiple HPVTypes

This example shows the performance of an embodiment of universalisothermal amplification referred to as “half uTMA”, in a system todetect at least 12 human papillomavirus (HPV) types associated with ahigh risk of developing cervical cancer (high-risk HPV types). Thetarget was either 200 or 1,000 copies/reaction (c/rxn) of a single invitro transcript of the specified HPV type. Target capture,amplification and probe detection by using hybridization protectionassay (HPA) which were all performed substantially as described earlier(U.S. Pat. Nos. 6,110,678 and 6,534,273 for target capture, U.S. Pat.Nos. 5,399,491 and 5,554,516 for TMA, and U.S. Pat. Nos. 5,283,174 and5,639,604 for HPV). The target capture mixture contained in the TCreagent 2 pmol each of target capture oligonucleotides of SEQ ID Nos.28-32. The target capture mixture additionally contained 5 pmol each ofHPV TSU T7 promoter primers of SEQ ID Nos. 1-9. Each of these primerscontained the target-specific region, the sequence of the universal T7primer, and a T7 promoter region. Amplification buffer containedreagents for performing TMA plus 15 pmol each of universal T7 primer ofSEQ ID NO:33 and the TS (target-specific) non-T7 primers of SEQ ID Nos.10-13.

During the target capture step, which includes hybridization at62.deg.C, the capture oligonucleotides and TSU T7 promoter primershybridized to their specific in vitro transcripts; and all unhybridizedprimers were removed during the wash steps. After target capture, themagnetic beads with bound complex that includes the target strand andhybridized TSU primer were mixed with amplification reagent containingprimers, RNA polymerase, reverse-transcriptase, dNTPs and NTPs, and thenincubated at 42.deg.C for 60 minutes. In the first step of the reaction(initial amplification phase), a cDNA transcription template is createdwhich incorporates the universal T7 primer region and a HPVtarget-specific binding region. Amplification proceeds (in the secondphase of amplification) by using the universal T7 promoter primer and anon-T7 primer specific for the target in the reaction. RNA ampliconswere detected by HPA by using a mixture of target-specific acridiniumester (AE)-labeled probes of SEQ ID Nos. 20-27. All probes nothybridized to an amplicon target were hydrolyzed by using the selectionreagent during the HPA procedure and rendered non-chemiluminescent.Probes that were bound to amplicon target and remained protected fromhydrolysis. HPA detection was performed by using the detection reagents,and the resulting chemiluminescent signals were measured and expressedin relative light units (RLU).

Table 1 shows RLU signals (average of 3 replicates) obtained for 12high-risk HPV types, 4 low-risk HPV types, and negative reactions inwhich no target was added. A positive reaction was scored for RLUgreater than 20,000. In this example, all high-risk HPV types weredetected successfully at 200 c/rxn, except HPV 45, which was positive at1,000 c/rxn. None of the low-risk HPV types tested gave a positivesignal.

TABLE 1 Group Target Avg RLU 200 c/rxn Avg RLU 1,000 c/rxn A1 HPV 163,125,124 3,335,360 HPV 31 345,676 1,524,821 HPV 35 2,948,726 3,207,962A2 HPV 33 2,571,697 3,924,319 HPV 58 922,123 4,270,230 C1 HPV 18 997,3561,438,953 HPV 45 12,839 579,850 HPV 59 1,950,796 2,521,835 C2 HPV 392,466,025 2,452,492 HPV 68 689,548 1,845,594 D HPV 51 1,571,8341,604,492 HPV 56 1,015,787 775,501 Avg 1 mil c/rxn Avg 10 mil c/rxnLow-risk types HPV 6  9,431 9,790 HPV 11 9,839 9,644 HPV 42 9,805 9,628HPV 43 9,683 9,714 Negative 7,612

Example 2: Sensitivity of Universal TMA System for Detection ofHigh-Risk HPV Types

This example shows the performance of an embodiment of universalisothermal amplification referred to as a “full uTMA” in a system thatincludes two universal sequences to detect 12 high-risk HPV virus types.The target was either 200 or 2,000 copies/reaction of a single in vitrotranscript of the specified HPV type. Target capture, amplification andHPA detection steps were all performed substantially as described inExample 1 except that different TSU primer combinations were used. Thetarget capture mixture contained 2 pmol each of TC oligonucleotides ofSEQ ID NOs. 28, 29, 30, 31 and 32. The target capture mixtureadditionally contained S-oligonucleotide TSU primer complexes designedto detect the 12 high-risk HPV types. The TSU primer complexes wereformed by hybridizing 5 pmol of TSU T7 promoter primer with 10 pmol ofS-oligonucleotide of SEQ ID NO:35 and 15 pmol of the corresponding TSUnon-T7 primer. The S-oligonucleotide primer complexes consisted of theS-oligonucleotide of SEQ ID NO:35 in hybridization complexes with thefollowing combinations of TSU T7 promoter primer plus TSU non-T7 primer:SEQ ID Nos. 1 plus 14, SEQ ID Nos. 2 plus 14, SEQ ID Nos. 3 plus 14 (thesame TSU non-T7 primer was used for 3 TSU T7 primers directed to arelated group of HPV types), SEQ ID Nos. 4 plus 15, SEQ ID Nos. 5 plus16, SEQ ID Nos. 6 plus 17, SEQ ID Nos. 7 plus 18, SEQ ID Nos. 8 plus 15,and SEQ ID Nos. 9 plus 15 (the same TSU non-T7 primer was used for bothTSU T7 primers directed to a related group of HPV types). Each TSU T7promoter primer contained the target-specific region, the sequence ofthe universal T7 primer, and a T7 promoter region. Each TSU non-T7primer contained the target-specific region and the sequence of theuniversal non-T7 primer. After each S-oligonucleotide primer complex wasformed separately, they were combined in the target capture mix.Amplification buffer contained 15 pmol of universal T7 promoter primerof SEQ ID NO:33 and universal non-T7 primer of SEQ ID NO:34.

During target capture hybridization at 62.deg.C, the captureoligonucleotides and TSU T7 promoter primers of the S-oligonucleotideprimer complexes hybridized to their specific in vitro transcripts; andall unhybridized primers and S-oligonucleotide primer complexes wereremoved during the wash steps. After target capture, the magnetic beadswith bound target/primer complexes were mixed with amplification reagentcontaining universal primers, RNA polymerase, reverse-transcriptase,dNTPs and NTPs, and then incubated at 42.deg.C for 60 minutes. In thefirst step of the amplification reaction a cDNA transcription templatewas created which incorporates the universal T7 primer region and auniversal non-T7 primer binding region and then amplification proceededby using the universal T7 and non-T7 primers. RNA amplicons weredetected by HPA as described above using a mixture of target-specificAE-labeled probes of SEQ ID Nos. 20 to 27. All probes not hybridized toan amplicon target were hydrolyzed during the HPA procedure and renderednon-chemiluminescent. Probes that were bound to amplicon target andremained protected. HPA detection was performed as described above, andthe resulting chemiluminescent signal was measured and expressed inrelative light units (RLU).

Table 2 shows signals (average of 3 replicates) obtained for 12high-risk HPV types, and negative reactions with no target added. Apositive reaction was scored for RLU greater than 20,000. In thisexample, all high-risk HPV types were detected successfully at 200c/rxn, except HPV 31, which was positive at 2,000 copies per reaction.In other experiments (data not shown), low-risk HPV types were notdetected.

TABLE 2 Group Target Avg RLU 200 c/rxn Avg RLU 2,000 c/rxn A1 HPV 1632,620 209,397 HPV 31 17,123 84,653 HPV 35 28,542 217,063 A2 HPV 3322,276 797,309 HPV 58 236,932 1,383,602 C1 HPV 18 103,672 964,766 HPV 45324,981 1,329,859 HPV 59 29,254 202,631 C2 HPV 39 100,941 1,376,088 HPV68 162,030 943,088 D HPV 51 241,543 1,132,808 HPV 56 447,408 483,658Negative 10,312

Example 3: Detection of HPV RNA from Clinical Samples Using a uTMASystem

This example shows that the “full uTMA” system as described in example 2is capable of detecting HPV RNA from cervical swab or scraping samplespreserved in alcohol-based liquid media (CYTYC™). The procedure wasperformed as described in Example 2, except that 100.micro.l of theliquid media sample was added to 500 μl of target capture mixture in thetarget capture reaction.

The presence of both high- and low-risk HPV was determined by HPV DNAPCR and visualized as bands following separation by agarose gelelectrophoresis. Identity of any HPV viral RNA present in the sampleswas confirmed by DNA sequencing. Samples that produced greater than20,000 RLU using the full uTMA system, were scored as positive. Table 3shows the correlation between HPV type and full uTMA amplificationresults. Positive PCR that resulted in highly visible bands were scoredas “+”, weak bands as “+/−”, and negative results (no visible band) as“−” (and “nd” means not determined). The full uTMA HPV system used inthis example was not optimized for sensitivity or specificity, butcorrectly scored 29 of 34 cervical samples in this study. Samples 6 and26 were probably not detected because of low amounts of HPV RNA.

TABLE 3 HPV type by Targeted Sample # PCR Result sequencing high-riskHPV uTMA result 1 + HPV 59 yes + 2 + HPV 16 yes + 3 +/− HPV 66 no − 4 +HPV 61 no − 5 + HPV 18 yes + 6 +/− HPV 18 yes − 7 + HPV 16 yes + 8 +mixed yes + 9 + 70 no − 10 + HPV 81 no − 11 + mixed yes + 12 + HPV 16yes + 13 + HPV 33 yes + 14 + HPV 58 yes + 15 + HPV 31 yes + 16 + HPV 18yes + 17 − nd nd − 18 − nd no − 19 + HPV 54 no − 20 − nd no − 21 − nd no− 22 − nd no − 23 + HPV 59 yes + 24 + HPV 16 yes + 25 + HPV 81 no − 26+/− HPV 68 yes − 27 + HPV 68 yes + 28 +/− HPV 53 no − 29 + HPV 16 yes +30 + HPV 62 no ++++ 31 + HPV 58 yes + 32 + HPV 16 yes + 33 + HPV 58yes + 34 + HPV 16 yes −

Example 4: Detection of PCA3 RNA in Uniplex and Multiplex Modes UsingReverse Standard TMA

In this example, reverse TMA was performed in a standard, i.e.,non-universal, format (RS-TMA). The assay was performed in either theuniplex mode, where the only oligonucleotides required for targetcapture, amplification and detection of PCA3 were included, or themultiplex mode, where oligonucleotides required for target capture,amplification and detection of both PCA3 and PSA were included. Theassay was performed substantially equivalently to the general protocoldescribed above. Specifically, PCA3 in vitro transcript (IVT; SEQ IDNO:62) was spiked into water/STM (1:1) at 10.sup.6, 10.sup.4 or 10.sup.2copies per reaction. For samples run in the uniplex mode, 5 pmol PCA3 TCprobe (SEQ ID NO:53), 2 pmol PCA3 blocker (SEQ ID NO:51), and 5 pmol ofPCA3 Non-T7 (NT7) primer (SEQ ID NO:49) were spiked into TCR, and 15pmol of PCA3 Non-T7 (NT7) primer (SEQ ID NO:49), 10 pmol of PCA3 T7promoter provider (SEQ ID NO:50) and 12 pmol PCA3 molecular torch (SEQID NO:52) were spiked into amplification reagent (amounts given here andlater in this and other examples are per reaction, unless indicatedotherwise). For samples run in the multiplex mode, in addition to thePCA3 oligomers listed above, 5 pmol PSA TC probe (SEQ ID NO:60), 2 pmolPSA blocker (SEQ ID NO:58) and 5 pmol of PSA NT7 primer (SEQ ID NO:56)were also spiked into TCR, and 15 pmol of PSA NT7 primer (SEQ ID NO:56),10 pmol of PSA T7 promoter provider (SEQ ID NO:57) and 12 pmol PSAmolecular torch (SEQ ID NO:59) were spiked into amplification reagent.For each sample, either 3 or 4 replicates were performed.

After the assay was completed, plots of fluorescence versus time wereprepared for each condition (FIG. 19) and average emergence times weredetermined (Table 4).

TABLE 4 Emergence time (min) PCA3 amount Uniplex Multiplex 10⁶ 8.5 12.510⁴ 11.5 >80 10² 14.5 >80

These results demonstrate that the RS-TMA readily detected PCA3 RNA in auniplex mode. However, in a multiplex mode (PSA specificoligonucleotides present in addition to the PCA3 specificoligonucleotides present in the uniplex mode), detection of PCA3 wasseverely hampered. In fact, 10.sup.2 and 10.sup.2 copies of PCA3 wereundetectable under the conditions of the assay. This illustrates theproblem that exists with multiplex amplification reactions known in theart.

These results further demonstrate the ability of RS-TMA to quantitatetarget level, as amount of PCA3 was directly related to the emergencetime. One drawback of the RS-TMA method is the small difference inemergence times between relatively large copy level differences of PCA3(i.e., 3 minutes difference in emergence time between 100-folddifferences in PCA3 copy level). This diminishes the ability of theRS-TMA method to accurately discriminate between small differences(e.g., 3-fold) in copy levels.

Example 5: Detection of PCA3 RNA in Uniplex and Multiplex Modes UsingReverse Universal (Half) TMA

In this example, reverse TMA was performed in a universal (half) TMAformat (RUh-TMA). In this format, a target-specific universal NT7 primer(TSU NT7) containing a specific target binding region and a universalregion at the 5′ end of the oligonucleotide is bound to target in thetarget capture step. Excess TSU-NT7 is washed away. A TSU-NT7 isincluded in the target capture step for each analyte to be detected in amultiplex assay. In the amplification reaction, a universal NT7 primer(same sequence as the universal sequence of all the TSU-NT7 primers) isadded and is used as the NT7 primer in the amplification of all theanalytes to be detected in a multiplex reaction. Also in theamplification reaction, a target specific T7 promoter provider (TS-T7)is added for each target to be detected in a multiplex assay. Aschematic representation of this format is given in FIG. 15.

The assay was performed substantially equivalently to the protocoldescribed in Example 4 above, with the exceptions described below.Specifically, a PCA3 TSU-NT7 primer (5 pmol; SEQ ID NO:48) and PSATSU-NT7 primer (5 pmol: SEQ ID NO:55) were spiked into TCR instead ofthe PCA3 and PSA TS-NT7 primers, respectively, cited in Example 4.Further, a universal NT7 primer (15 pmol; SEQ ID NO:64) was spiked intothe amplification reaction instead of the PCA3 TS-NT7 primer in theuniplex mode and instead of both the PCA3 and PSA TS-NT7 primers in themultiplex mode. All other conditions were the same as those given inExample 5. After the assay was completed, average emergence times weredetermined (Table 5).

TABLE 5 Emergence time (min) Uniplex Multiplex PCA3 amount RS-TMARUh-TMA RS-TMA RUh-TMA 10⁶ 7.0 8.0 11.5 9.5 10⁴ 10.0 12.0 >80 11.5 10²14.0 17.5 >80 24.0

These results demonstrate that the RUh-TMA format readily detected PCA3RNA. In the uniplex mode, emergence times are somewhat later than thecorresponding emergence times obtained with the RS-TMA format. This isfavorable in relation to quantitation, and helps to solve the problemwith RS-TMA cited in Example 4 (i.e., diminished ability of the RS-TMAmethod to accurately discriminate between small differences (e.g.,3-fold) in copy levels). In the multiplex mode, the interferencesobserved in the RS-TMA system are largely overcome, resulting in readydetection of all levels of PCA3 RNA tested.

Example 6: Detection of PCA3 RNA in Uniplex and Multiplex Modes UsingReverse Universal (Full) TMA (RUf-TMA) in the S-Oligo Format

In this example, reverse TMA was performed in a universal (full) TMAformat (RUh-TMA). In universal (full) TMA, amplification is initiatedwith a TSU-NT7 and a TSU-T7 provider, and a universal NT7 primer and auniversal T7 provider drive subsequent rounds of amplification. In orderto provide each target with the primer and provider required forinitiation, yet include only a universal primer and provider in theamplification reaction, a TSU NT7 primer and a TSU T7 provider arejoined together, this complex is bound to target in the target capturestep (via hybridization of the target specific region of the TSU-NT7 tothe target) and excess complex is washed away. In amplification, theTSU-NT7 primer is extended, and after digestion of the target via RNAseH, the target specific region of the TSU-T7 provider that is joined tothe TSU-NT7 primer binds to the cDNA and amplification is initiated.Amplification then continues using the universal NT7 primer and T7provider that are in the amplification reagent.

In the S-oligo mode of RUf-TMA described in this example, the TSU-NT7primer and TSU-T7 provider are joined via hybridization of both to anintervening “S-oligo” as shown schematically in FIG. 16. This S-oligocomplex is pre-formed for each analyte to be included in a multiplexassay, then all are added to TCR in the manner that NT7 primers areadded in the RS- and RUh-TMA formats described above.

The assay in this example was performed substantially equivalently tothe protocol described in Example 4 above, with the exceptions describedbelow. Specifically, the multiplex portion of the assay contained theoligonucleotides required for target capture, universal amplificationand real time detection of not only PCA3 and PSA, but also AMACR. PCA3S-oligo complex was prepared by mixing 5 pmol of PCA3 TSU-NT7 primer(SEQ ID NO:48), 7.5 pmol S-oligo (SEQ ID NO:66) and 10 pmol PCA3 TSU-T7provider (SEQ ID NO:50; in this case, the TS- and TSU-T7 providers areone and the same in water/STM/TCR (1/1/0.5). Further, PSA S-oligocomplex was prepared by mixing 5 pmol of PSA TSU-NT7 primer (SEQ IDNO:55), 7.5 pmol S-oligo (SEQ ID NO:66) and 10 pmol PSA TSU-T7 provider(SEQ ID NO:57). AMACR S-oligo complex was prepared by mixing 5 pmol ofAMACR TSU-NT7 primer (SEQ ID NO:36), 7.5 pmol S-oligo (SEQ ID NO:66) and10 pmol AMACR TSU-T7 provider (SEQ ID NO:37). The mixtures wereincubated at room temperature for 30 minutes to allow the complexes toform. PCA3 and PSA TC probes and blockers were spiked into TCR as inExample 5. Additionally, AMACR TC probe (5 pmol; SEQ ID NO:40) and AMACRblocker (2 pmol; SEQ ID NO:38) were also spiked into TCR. PCA3 and PSAS-oligo complexes (5 pmol each) were spiked into TCR instead of PCA3 andPSA TS-NT7 primers, respectively. AMACR S-oligo complex (5 pmol) wasalso spiked into TCR. PCA3 and PSA molecular torches were spiked intoamplification reagent as in Example 5. Additionally, AMACR moleculartorch (12 pmol; SEQ ID NO:39) was also spiked into amplificationreagent. Universal NT7 primer (15 pmol; SEQ ID NO:64) and universal T7provider (10 pmol; SEQ ID NO:65) were spiked into the amplificationreagent instead of the TS-NT7 primer(s) and TS-T7 provider(s). All otherconditions were the same as those given in Example 4.

After the assay was completed, average emergence times were determined(Table 6).

TABLE 6 Emergence time (min) PCA3 amount Uniplex Multiplex 10⁶ 18.1 20.210⁴ 23.4 25.4 10² 34.5 36.5

These results demonstrate that the RUf-TMA format in the S-oligo modereadily detected PCA3 RNA. In the uniplex mode, emergence times aresignificantly later and the time between different copy levels issignificantly greater than the corresponding values obtained with theRS-TMA format. These features are very favorable in relation toquantitation, and help to solve the problem with RS-TMA cited in Example5 (i.e., diminished ability of the RS-TMA method to accuratelydiscriminate between small differences (e.g., 3-fold) in copy levels).In the multiplex mode, the interferences observed in the RS-TMA systemare largely overcome, resulting in ready detection of all levels of PCA3RNA tested.

Example 7: Detection of PCA3 RNA in Uniplex and Multiplex Modes

In this example, reverse TMA was performed in a universal (full) TMAformat (RUf-TMA) very similar to that described in Example 6. However,instead of via an S-oligo complex, TSU NT7 primer and TSU T7 providerwere joined together using a Directly Hybridized-oligo (DH-oligo)complex. In this mode, the TSU NT7 primer and TSU T7 provider aredirectly hybridized to one another, with no intervening sequence as inthe S-oligo complex. FIG. 17 depicts an example of a DH-oligo complex,in this case with binding occurring via the T7 promoter region of the T7provider.

The assay in this example was performed substantially equivalently tothe protocol described in Example 6, with the exceptions describedbelow. Specifically, PCA3 DH-oligo complex was prepared by mixing 5 pmolof PCA3 DH-TSU-NT7 primer (SEQ ID NO:54) and 5 pmol PCA3 TSU-T7 provider(SEQ ID NO:50) in water/STM/TCR (1/1/0.5). Further, PSA DH-oligo complexwas prepared by mixing 5 pmol of PSA DH-TSU-NT7 primer (SEQ ID NO:61)and 5 pmol PSA TSU-T7 provider (SEQ ID NO:57). The mixtures wereincubated at room temperature for 30 minutes to allow the complexes toform. TC probes and blockers were spiked into TCR as in Example 6, butPCA3 and PSA DH-oligo complexes (5 pmol each) were spiked into TCRinstead PCA3 and PSA S-oligo complexes, respectively. All otherconditions were the same as those given in Example 6, except that thetotal amplification volume was 0.04 mL instead of 0.08 mL (0.03 mLamplification reagent and 0.01 mL enzyme reagent). After the assay wascompleted, average emergence times were determined (Table 7).

TABLE 7 Emergence time (min) PCA3 amount Uniplex Multiplex 5 × 10⁶ 49.550.5 5 × 10⁵ 43.0 44.0 5 × 10⁴ 36.5 37.5 5 × 10³ 30.0 31.0 5 × 10² 24.524.5

These results demonstrate that the RUf-TMA format in the DH-oligo modereadily detected PCA3 RNA. In the uniplex mode, emergence times aresignificantly later and the time between different copy levels issignificantly greater than the corresponding values obtained with theRS-TMA format. These features are very favorable in relation toquantitation, and help to solve the problem with RS-TMA cited in Example4 (i.e., diminished ability of the RS-TMA method to accuratelydiscriminate between small differences (e.g., 3-fold) in copy levels).In the multiplex mode, the interferences observed in the RS-TMA systemare largely overcome, resulting in ready detection of all levels of PCA3RNA tested. Plots of emergence time versus PCA3 copy levels for both theuniplex and multiplex assays yielded excellent correlation factors(uniplex R.sup.2=1.000; duplex R.sup.2=1.000), demonstrating thequantitative nature of these assays.

Example 8: Detection of PCA3 RNA in Uniplex and Multiplex Modes UsingReverse Universal (Full) TMA (RUf-TMA) in the CL-Oligo Format

In this example, reverse TMA was performed in a universal (full) TMAformat (RUf-TMA) very similar to that described in Example 6. However,instead of via an S-oligo complex, TSU NT7 primer and TSU T7 providerwere joined together using a covalently linked-oligo (CL-oligo) complex.In this mode, the TSU NT7 primer and TSU T7 provider are covalentlylinked to one another at the 5′-ends of each oligomer. A variety ofmethods can be utilized to achieve such a linking. An example of onepossible scheme is shown schematically in FIG. 18. In this case, the NT7primer and T7 provider are joined 5′ to 5′ with 2 C9 linkers between the2 oligomers.

The assay in this example was performed substantially equivalently tothe protocol described in Example 6 above, with the exceptions describedbelow. Specifically, the multiplex portion of the assay contained theoligonucleotides required for target capture, universal amplificationand real time detection of not only PCA3 and PSA, but also AMACR andCAP2. CL-oligos for each analyte were prepared generally as follows: NT7primers and T7 providers were synthesized using standard phosphoramiditereagents (Sigma Aldrich), except for those listed below, using anExpedite DNA synthesizer (Applied Biosystems, Foster City, Calif.). TheT7 provider was synthesized with a 5′-aldehyde (specialtyphosphoramidite from SoluLink, San Diego, Calif.) and a reverse polaritydC (specialty Control Pore Glass (CPG) reagent from BiosearchTechnologies). The NT7 primer was synthesized with a 5′ C6 amino linker(Glen-Research). Both oligos underwent cleavage and deprotection usingstandard conditions. A bifunctional spacer was then attached to the NT7primer via incubation with Hydrazine-NHS ester (SoluLink) at roomtemperature for 2 hours in 100 mM phosphate buffer (pH 7.40) containing150 mM NaCl. The reaction mixture was then precipitated with sodiumacetate (pH 5.1) and the pellet was dissolved in 100 mM MOPS buffer (pH4.8) containing a 10% excess of the 5′aldehyde-modified T7 provider.This mixture was left overnight at room temperature and subsequentlydesalted and purified by PAGE.

SEQ ID numbers of oligonucleotides used to construct the CL-oligocomplexes are in Table 8

TABLE 8 Oligo Analyte Type SEQ ID No PCA3 TSU NT7 primer 48 TSU T7provider 50 PSA TSU NT7 primer 55 TSU T7 provider 57 AMACR TSU NT7primer 36 TSU T7 provider 37 CAP2 TSU NT7 primer 42 TSU T7 provider 43PCA3 and PSA TC probes and blockers were spiked into TCR as in Example7, but PCA3 and PSA DH-oligo complexes were replaced with PCA3 and PSACL-oligo complexes (5 pmol each), respectively. Additionally, AMACR TCprobe (5 pmol; SEQ ID NO:40), AMACR blocker (2 pmol, SEQ ID NO:38), CAP2TC probe (5 pmol; SEQ ID NO:46) and CAP2 blocker (2 pmol, SEQ ID NO:44)were also spiked into TCR. Further, in addition to the oligonucleotideslisted in Example 7, AMACR molecular torch (12 pmol; SEQ ID NO:39) andCAP2 molecular torch (12 pmol; SEQ ID NO:45) were also spiked into theamplification reagent. All other conditions were the same as those givenin Example 7. After the assay was completed, average emergence timeswere determined (Table 9).

TABLE 9 Emergence time (min) PCA3 amount Uniplex Multiplex 10⁶ 35.0 35.510⁴ 49.0 48.5 10² 59.0 59.5

These results demonstrate that the RUf-TMA format in the CL-oligo modereadily detected PCA3 RNA. In the uniplex mode, emergence times aresignificantly later and the time between different copy levels issignificantly greater than the corresponding values obtained with theRS-TMA format. These features are very favorable in relation toquantitation, and help to solve the problem with RS-TMA cited in Example5 (i.e., diminished ability of the RS-TMA method to accuratelydiscriminate between small differences (e.g., 3-fold) in copy levels).In the multiplex mode (quadruplex in this example), the interferencesobserved in the RS-TMA system are largely overcome, resulting in readydetection of all levels of PCA3 RNA tested.

Example 9: Detection of PCA3, PSA, AMACR and CAP2 in Uniplex andMultiplex Modes Using Reverse Universal (Three-Quarters) TMA (RUt-TMA)

In this example, uniplex samples were made in quadruplicate to contain2E6, 2E5, 2E4, 2E3 or 2E2 copies of PCA3, PSA, AMACR and/or CAP2 targetnucleic acid. No target nucleic acids were added to the negativecontrol. For the uniplex reactions, a series of target capture reagentsare prepared as is generally described herein. For the reverse universalthree-quarters reactions, each of these target capture reagentscomprises a target capture oligomer, about 5 pmoles of a TSU Non-T7primer and a blocker oligomer as follows: for the PCA3 reaction SEQ IDNOS:48, 53 & 51; for the PSA reaction SEQ ID NOS:60, 58 & 55; for theAMACR reaction SEQ ID NOS:40, 38 & 36 and for the CAP2 reaction SEQ IDNOS:46, 44 & 42. Target capture was performed at 60.deg.C for about 30minutes followed by an incubation at room temperature for about 30minutes. The captured target nucleic acids were washed to remove,amongst other things, unhybridized TSU Non-T7 primers and blockeroligomers. The capture target nucleic acids were isolated and thentransferred and resuspended into an amplification reagent comprisingabout 0.07 pmoles per reaction of a TSU-T7 oligomer, about 15 pmoles perreaction of a universal T7 oligomer and about 15 pmoles per reaction ofa universal non-T7 oligomer. The universal primers target the complementof the universal sequences introduced into the amplification product bythe TSU Non-T7 and the TSU T7 amplification oligomers. The TSU T7oligomer targets the cDNA strand generated by the TSU Non-T7. RNAtranscripts generated from this promoter provider comprise the universalsequence and the target specific sequence of the TSU T7. Subsequentrounds of amplification use either of the TSU T7 or the universal T7,until the TSU T7 amounts are exhausted. Amplification was performed at42.deg.C for 80 minutes. Detection was in real time using a moleculartorch (SEQ ID NOS:52, 59, 39, 45 for PCA3, PSA, AMACR and CAP2,respectively). Uniplex Rut-TMA for each of the analytes tested performedexceptionally well. Results are as follows (copy number/averageemergence time): for PCA3 at 2E2/54 minutes, 2E3/46 minutes, 2E4/40minutes, 2E5/36 minutes and 2E6/32 minutes; for PSA at 2E2/63 minutes,2E3/56 minutes, 2E4/48 minutes, 2E5/43 minutes and 2E6/37 minutes; forAMACR at 2E2/62 minutes, 2E4/42 minutes and 2E6/34 minutes; and CAP2 at2E2/50 minutes, 2E4/41 minutes and 2E6/34 minutes.

Similarly, an oligomer multiplex reaction was performed, wherein each ofthe samples contained, in quadruplicate, 2E6, 2E5, 2E4, 2E3 and/or 2E2copies of one of the following target nucleic acids: PCA3, PSA, AMACR orCAP2, and all of the oligomers required to support target capture,amplification and detection of each of the other analytes listed(quadruplex oligos). Negative control was sample transport medium alone.A target capture reagent was prepared comprising target captureoligomers, blockers and 5 pmoles per reaction of each TSU Non-T7oligomer for all four of the targets (SEQ ID NOS:53, 51, 48, 60, 58, 55,40, 38, 36, 46, 44 & 43). Target capture and wash was performed asdescribed directly above, and the captured target nucleic acids weretransferred and resuspended into an amplification reagent. Amplificationreagent comprised about 0.07 pmoles per reaction of each of the TSU T7(SEQ ID NOS:48, 55, 36 & 42). The amplification reagent furthercomprised universal T7 oligomers (SEQ ID NO:65) and universal non-T7oligomers (SEQ ID NO:64). Amplification was performed at 42.deg.C for 30minutes. Detection was in real time using a molecular torch (SEQ IDNOS:52, 59, 39 & 45 for PCA3, PSA, AMACR and CAP2, respectively). Themultiplex Rut-TMA format for each of the analytes tested performed well.Emergence times were very similar to those obtained in the uniplexreactions discussed directly above, thereby demonstrating that theRut-TMA format overcomes the potential adverse reaction betweenamplification oligomer for different target nucleic acids in a multiplexreaction. Results are as follows (copy number/average emergence time):for PCA3 at 2E2/58 minutes, 2E3/49 minutes, 2E4/44 minutes, 2E5/40minutes and 2E6/34 minutes; for PSA at 2E2/66 minutes, 2E3/57 minutes,2E4/47 minutes, 2E5/42 minutes and 2E6/36 minutes; for AMACR at 2E2/54minutes, 2E4/44 minutes and 2E6/34 minutes; and CAP2 at 2E2/55 minutes,2E4/46 minutes and 2E6/36 minutes.

Example 10: Detection of PCA3 and AMACR in Uniplex and Multiplex ModesUsing Reverse Universal (Quarter) TMA (RUq-TMA) and Reverse Universal(Two-Quarter) TMA (RUqq-TMA)

In RUq-TMA, the amplification oligomers comprise only one TSU oligomer.Thus, the initial amplification product contains only one universalsequence and subsequent amplification is performed with a universalamplification oligomer and a target specific oligomer. The TSU oligomercan be either the T7 or the Non-T7 amplification oligomer. In RUqq-TMA,two TSU oligomers are used; a TSU T7 and a TSU Non-T7. The RUq and RUqqreactions eliminate the need for a heated target capture step when theTSU oligomers are provided as part of the amplification reagent. Targetcapture can be performed using a wobble probe, which does not requireheat. By eliminating the high heat requirements of specific targetcapture and TSU complex binding in the target capture step, thisamplification assay becomes useful in situations where a high heatsource is either unavailable or undesired.

a. RUcw-TMA Using Specific Target Capture Plus Higher Heat orNon-Specific Target Capture and Lower Heat.

In a first example, a series of reactions were prepared foramplification and detection of PCA3 using either a target capturereagent comprising a wobble target capture oligomer (SEQ ID NO:113) or atarget specific target capture oligomer (SEQ ID NO:53). Samples wereprepared to contain 1E6, 1E4 or 300 copies of PCA3 target nucleic acid.Negative controls were sample transport media without added sample.Target capture reagents were prepared to comprise either SEQ ID NO:113or SEQ ID NO:53, which are the wobble oligomer or the target specificoligomer, respectively. Each of the target capture reagents was added toa series of samples in quadruplicate. Target capture for the series ofsamples using SEQ ID NO:53 was performed as is generally described:incubate at 60.deg.C for about 30 minutes; incubate at room temperaturefor about 30 min and wash. Target capture for the series of samplesusing SEQ ID NO:113 was performed as follows: incubate at roomtemperature for 20 min and wash (see e.g., WO 2008/016988 for adescription of target capture using a wobble target capture oligomer).Captured samples were then resuspended into amplification reactionmixtures.

Amplification reaction mixtures comprised 0.07 pmoles per reaction of aTSU Non-T7 amplification oligomer (SEQ ID NO:48), about 0.07 pmoles perreaction of TSU T7 amplification oligomers (SEQ ID NO:50), about 0.5pmoles of blocker oligomer (SEQ ID NO:51), about 15 pmoles per reactionof a universal Non-T7 amplification oligomer (SEQ ID NO:64), about 15pmoles per reaction of a universal T7 amplification oligomer (SEQ IDNO:65) and about 10 pmoles of a molecular torch (SEQ ID NO:52).Amplification was performed at 42.deg.C, and fluorescence was monitoredthroughout amplification. Overall for this example, both target specificand non-specific target capture performed well, with the target specificoligomer yielding somewhat better performance that did the wobbleoligomer. For 1E6 copies of PCA3 target nucleic acid, the targetspecific capture had an average emergence time of 30.5 minutes, whilethe wobble oligomer capture had an average emergence time of 32.2minutes. Similarly for 1E4 and 300 copies of PCA3, target specificcapture emergence times were 37.5 minutes and 44.4 minutes, whilenon-specific wobble oligomer capture was 40.8 minutes and 49.5 minutes.These results show that the RUqq-TMA system works well in amplificationand detection systems wherein higher heat is not desired, not feasibleor not acceptable.

b. RUq-TMA Using a TSU Non-T7 Amplification Oligomer.

Uniplex amplification assays were run using TSU Non-T7 amplificationoligomers. Samples comprised 300 copies of PCA3. Negative controls weresample transport medium without addition of sample nucleic acid. Targetcapture reagents were prepared to comprise 5 pmoles per reaction oftarget capture oligomer (SEQ ID NO:53) and, optionally, 2 pmoles perreaction of blocker oligomer (SEQ ID NO:51). A series of amplificationreagents were prepared to comprise TSU Non-T7 amplification oligomers(SEQ ID NO:48) at one of 1, 0.5 or 0.05 pmoles per reaction, a universalNon-T7 amplification oligomer (SEQ ID NO:64) at about 15 pmoles perreaction and about 10 pmoles per reaction of target specific T7amplification oligomer (SEQ ID NO:50). Optionally, blocker oligomers canbe provided in the amplification reagent at 0.5 pmoles per reaction.Preferably, blocker oligomer is provided in the reaction. Here, theblocker was present in the amplification reagent. Universal T7 oligomerswere not added into the amplification reagent, thus the T7 side of thereaction is target specific. Target capture was performed on the samplepreparations as is generally described herein, and captured targets wereresuspended into amplification reaction mixtures. Detection wasperformed throughout these amplifications using a molecular torchtargeting PCA3 amplification product (SEQ ID NO:52). In this example,the results showed that PCA3 amplified well at 300 copies using thisRUq-TMA format. Average emergence times were 16.5 minutes for theamplification reaction using 1 pmole per reaction of TSU Non-T7amplification oligomers; 17 minutes when using 0.5 pmoles per reactionand 20 minutes when using 0.05 pmoles per reaction.

c. RUq-TMA Using a TSU T7 Amplification Oligomer.

Uniplex amplification assays were run using TSU T7 amplificationoligomers. Samples comprised 1E6, 1E4 or 300 copies of PCA3. Negativecontrols were sample transport medium without addition of sample nucleicacid. Target capture reagents were prepared to comprise 5 pmoles perreaction of target capture oligomer (SEQ ID NO:53). A series ofamplification reagents were prepared to comprise one of 0.2, 0.1 or 0.05pmoles per reaction of TSU T7 amplification oligomers (SEQ ID NO:50), 5pmoles per reaction of blocker oligomer (SEQ ID NO:51), 15 pmoles perreaction of universal T7 amplification oligomer (SEQ ID NO:65) and 15pmoles per reaction of TSU Non-T7 amplification oligomer (SEQ ID NO:48).As above, blocker could optionally be provided in the target capturereagent, though here it was provided in the amplification reagent.Universal Non-T7 amplification oligomers were not added into theamplification reagent, thus the Non-T7 side of this amplificationreaction was target specific. Target capture was performed as isgenerally described herein, and captured targets were resuspended intoamplification reaction mixtures. Detection was performed throughoutthese amplifications using a molecular torch targeting PCA3amplification product (SEQ ID NO:52). In this example, the resultsshowed that PCA3 amplified well at 300 copies using this RUq-TMA format,with emergence times of 49, 56 and 63 minutes for TSU T7 amounts of 0.2,0.1 and 0.05 pmoles per reaction, respectively.

d. RUqq-TMA in Duplex Mode.

Samples were prepared as follows: 1E6 copies of PCA3 target nucleic acidand 1E6 copies of AMACR target nucleic acid, 1E4 copies of PCA3 targetnucleic acid and 1E4 copies of AMACR target nucleic acid, and 300 copiesof PCA3 target nucleic acid and 300 copies of AMACR target nucleic acid.Target capture reagents comprised target capture oligomers (SEQ IDNOS:40 & 53). Samples and target capture reagents were combined and thena target capture/wash procedure was performed generally as is describedherein. Captured targets were resuspended into amplification reactionmixture. The amplification reactions mixtures comprised blockeroligomers (SEQ ID NOS:38 & 51, each at 0.3 pmoles per reaction);TSU-Non-T7 amplification oligomer (SEQ ID NOS:48 & 36, each at 0.01pmoles per reaction), TSU-T7 amplification oligomers (0.15 pmoles perreaction of SEQ ID NO:50 & 0.25 pmoles per reaction of SEQ ID NO:37);universal Non-T7 amplification oligomers (SEQ ID NO:64, at 15 pmoles perreaction) and universal T7 amplification oligomer (SEQ ID NO:65 at 15pmoles per reaction). Detection was performed throughout theseamplifications using molecular torches targeting PCA3 amplificationproduct and AMACR amplification product (SEQ ID NOS:52 & 39,respectively).

In this example, samples containing both PCA3 and AMACR amplified well,demonstrating that the RUqq-TMA format is effective in multiplexreactions. Results are as follows: for 1E6 PCA3 and 1E6 AMACR, PCA3emerged at 32 minutes on average and AMACR emerged at 33 minutes onaverage; for 1E4 PCA3 and 1E4 AMACR, PCA3 emerged at 41 minutes onaverage and AMACR emerged at 42 minutes on average; and for 300 PCA3 and300 AMACR, PCA3 emerged at 48 minutes on average and AMACR emerged at 48minutes on average.

Example 11: Detection of PSA Using Reverse Universal (Half) Switched TMA(RUh-Switched-TMA)

Samples comprised 1E4, 1E3 and 1E2 copies of PSA. Negative controls weresample transport medium without addition of sample nucleic acid. Targetcapture reagents were prepared to comprise 5 pmoles per reaction oftarget capture oligomer (SEQ ID NO:60), 5 pmoles per reaction blocker(SEQ ID NO:58), and 5 pmol of a DH complex comprising equal amounts ofPSA TS-Non-T7-cPRO (SEQ ID NO:114) and PSA TSU-T7-T15 (SEQ ID NO:115).Amplification reagents were prepared to comprise 10 pmoles per reactionPSA TS-Non-T7 (SEQ ID NO:56) and 5 pmoles per reaction universal T7-T15(SEQ ID NO:116). Target capture was performed as is generally describedherein, and captured targets were resuspended into amplificationreaction mixtures. Detection was performed throughout theseamplifications using a molecular torch targeting PSA amplificationproduct (SEQ ID NO:59). In this example, the results showed that PSAamplified well at all target levels tested using the RUh-switched-TMAformat, with emergence times of 31, 35 and 40 minutes for 1E4, 1E3 and1E2 copies per reaction of PSA target, respectively.

Example 12: Uniplex Pre-Amplification, Split Secondary Amplification andDetection of PCA3 Using a TSU-Complex with a DH Linkage

Examples 12, 13 and 14 show that a linear pre-amplification method usinglinked forward and reverse primers (such as DH-complexes) is effectivein increasing the amount of product from the input target, which canthen be further amplified in a secondary exponential amplificationreaction. Further, pre-amplification of multiple targets in the samereaction had no adverse effect on accurate quantification of any of thetargets in the separated specific exponential amplification reactions.Because the pre-amplification step forms a specific cDNA from each inputtarget, which is then transcribed by an RNA polymerase in the samereaction in a linear manner, interference from other targets and theirprimers was not observed. In each example the DH-complexes were formedprior to being added to the target capture reagent. Thepre-amplification and the amplification steps were then performedsubstantially as is described

In this example, a first linear pre-amplification reaction was performedon a sample containing 1E6, 1E4 or 1E2 copies of PCA3 target nucleicacid. Following pre-amplification, part of the sample was transferred toan amplification reaction comprising target specific amplificationoligomers for the PCA3 target nucleic acid. Because the secondaryamplification uses target specific oligomers rather than universalamplification oligomers, the TSU-complexes provided in the preamplification are not used for their universal sequences. Thus, thesecomplexes can also be referred to as DH-complexes, which is the linkagemechanism used in this example. Also notably, this example is notlimited to the DH linkage format, as other direct or indirect linkagesmay be used as well.

A single target capture reagent was prepared to comprise SEQ ID NOS:81,69, 87 & 75 (5 pmoles each per reaction) as a target capture oligomers,SEQ ID NOS:82, 70, 88 & 16 (5 pmoles each per reaction) as a blockeroligomers and oligomers for four different DH-complexes: SEQ ID NOS:83 &84 (5 pmoles:7.5 pmoles per reaction), SEQ ID NOS: 71 & 72 (5 pmoles:7.5pmoles per reaction), SEQ ID NOS: 89 & 90 (5 pmoles:7.5 pmoles perreaction) and SEQ ID NOS: 77 & 78 (5 pmoles:7.5 pmoles per reaction).Thus, the target capture reagent provided target capture oligomers,blocker oligomers and DH-complex oligomers for PCA3 and for AMACR, PSAand CAP2. Only PCA3 was present in the samples. Target capture reagentwas combined with sample or with sample transport media alone as anegative control. A target capture protocol was performed as isgenerally described herein. Briefly, target capture proceeded at60.deg.C for 30 minutes followed by incubation at room temperature for20 minutes. Captured target with hybridized DH-complex was then washedtwice using a magnetic bead capture system, e.g., a KingFisher magneticbead capture system. Following capture and wash, the captured target andDH-complexes are transferred and resuspended into a pre-amplificationreaction. The pre-amplification reaction comprised a reversetranscriptase and RNA polymerase, and the pre-amplification step wasperformed at 42.deg.C for 15 minutes. No primers are added into thepre-amplification reaction, thus only the DH-complex hybridized totarget is present in the pre-amplification. Pre-amplification producesRNA transcripts from a cDNA produced by the target and DH-complex.

Following pre-amplification, four separate aliquots of thepre-amplification product each individually added to one of fourseparate secondary amplification reactions, each comprising a RNApolymerase, a reverse transcriptase, a different set of target specificoligomers and a different molecular torch for real-time detection ofsecondary amplification product: PCA3 specific secondary amplificationreaction comprised SEQ ID NOS:84 (10 pmoles), 85 (15 pmoles) & 86 (12pmoles); AMACR specific secondary amplification reaction comprised SEQID NOS:72 (10 pmoles), 73 (15 pmoles) & 74 (12 pmoles); PSA specificsecondary amplification reaction comprised SEQ ID NOS:90 (10 pmoles), 91(15 pmoles) & 92 (12 pmoles); and CAP2 specific secondary amplificationreaction comprised SEQ ID NOS:78 (10 pmoles), 79 (15 pmoles) & 80 (12pmoles). The secondary amplification was performed at 42.deg.C for 80minutes and the generation of amplification product was monitoredthroughout using the molecular torches. Results for this experimentshowed no false positives in the negative control samples. Also,secondary amplification reactions comprising target specific oligomersfor AMACR, PSA or CAP2 were also negative. The secondary amplificationreaction for PCA3 showed good sensitivity down to 100 copies of targetnucleic acid in the pre-amplification reaction. PCA3 results are asfollows (n=4): 1E6 copies in pre-amplification showed a secondaryamplification average emergence time of about 23 minutes; 1E4 copies inpre-amplification showed a secondary amplification average emergencetime of about 32 minutes; and 1E2 copies in pre-amplification showed asecondary amplification average emergence time of about 47 minutes.Using the DH-complexes in a multiplex amplification reaction providesgood amplification results for specific targets. Following the targetcapture, unhybridized oligomers and others undesired components in thesample, can be washed away, leaving captured target hybridized with aDH-complex. The pre-amplification reaction then performs well becausethe abundance of interfering oligomers was substantially reduced oreliminated. Primer dimers, spurious product formation, mis-priming atnon-target sequences and other common multiplex problems aresubstantially reduced or eliminated, as well. Pre-amplification isperformed using a reverse transcriptase to generate a cDNA, from theDH-complex, then, following binding to the T7 member of the DH-complexto the cDNA, an abundance of RNA transcripts are produced therefrom.Pre-amplified sample is transferred to one or more target specificsamples, each of which comprises the oligomers for only a single target.

Example 13: Multiplex Pre-Amplification, Split and Detection of PSA andPCA3 Using a TSU Complex in the Pre-Amplification Reaction and UniversalAmplification Oligomers in the Secondary Amplification Reaction or Usinga DH-Complex in the Pre-Amplification Reaction and Target SpecificAmplification Oligomers in the Secondary Amplification Reaction

This example performed target capture and pre-amplification on samplescontaining PCA3, PSA or PCA3 and PSA target nucleic acids usingTSU-complexes and DH-complexes targeting those nucleic acids. Followingpre-amplification, the reactions were split into secondary reactionscomprising either target specific secondary amplification oligomers oruniversal amplification oligomers. Secondary amplification reactionswere performed in the presence of a molecular torch for real timedetection of amplification product. The target specific secondaryamplification was superior to the universal secondary amplification. Asmentioned above, amplification oligomer-complexes can compriseamplification oligomer members, wherein one or both of the oligomermembers contain a universal tag sequence (TSU-complex) or whereinneither oligomer member contains a universal tag sequence (DH-complex).Furthermore, for these examples, if an oligomer member comprises auniversal tag sequence, but secondary amplification is performed usingamplification oligomer that are target specific, the complex is referredto as a DH-complex because universal amplification oligomers were notused in the secondary amplification.

Samples were prepared to comprise either 0 copies of PCA3 target nucleicacid and 1E3 copies of PSA target nucleic acid; 1E3 copies of PCA3target nucleic acid and 0 copies of PSA target nucleic acid; 1E5 copiesof PCA3 target nucleic acid and 1E3 copies of PSA target nucleic acid;or 1E3 copies of PCA3 target nucleic acid and 1E5 copies of PSA targetnucleic acid. Negative controls contained no added target nucleic acid.Target capture reagent was prepared to comprise target captureoligomers, TSU/DH complexes and blocker oligomers for each targetnucleic acid, (for PCA3, 5 pmoles/rxn SEQ ID NO:81, 5 pmoles/rxn SEQ IDNO:82, 5 pmoles/rxn SEQ ID NO:83 & 7.5 pmoles/rxn SEQ ID NO:84; and forPSA, 5 pmoles/rxn SEQ ID NO:87, 5 pmoles/rxn SEQ ID NO:88, 5 pmoles/rxnSEQ ID NO:89 & 7.5 pmoles/rxn SEQ ID NO:90). Target capture reagent wasadded to each sample. Target capture and wash was performed as isgenerally described herein. Following target capture and wash, thecaptured samples were transferred and resuspended into apre-amplification reagent comprising reverse transcriptase and RNApolymerase. Pre-amplification reaction was performed at 42.deg.C for 15minutes.

Following pre-amplification, the samples were then split into separatesecondary amplification reactions comprising RNA polymerase, reversetranscriptase, and one of the following sets of secondary amplificationand detection oligomers: (PCA3 target specific was 10 pmoles SEQ IDNO:84, 15 pmoles SEQ ID NO:85 and 12 pmoles SEQ ID NO:86; PSA targetspecific was 10 pmoles SEQ ID NO:90, 15 pmoles SEQ ID NO:91 and 12pmoles SEQ ID NO:92; PCA3 universal was 10 pmoles SEQ ID NO:93, 15pmoles SEQ ID NO:94 and 12 pmoles SEQ ID NO:86; and PSA universal was 10pmoles SEQ ID NO:93, 15 pmoles SEQ ID NO:94 and 12 pmoles SEQ ID NO:92).Amplification was performed at 42.deg.C for 80 minutes. Results for thisexample showed good amplification of each target using the targetspecific oligomers in the secondary amplification. Target specificamplification of one target nucleic acid in the presence of an excess ofanother target nucleic acid, 100-fold excess in this example, alsoprovided good results. Because the TSU-complexes confer universal primerbinding regions to the pre-amplified targets, the secondaryamplification reaction used universal primers. Thus, in the secondaryamplification reaction, all targets competed for the universal primers.However, using the target-specific primers instead, only the specifictarget in each reaction was amplified. As is seen by the below results,the secondary amplification reactions using target specificamplification oligomers had average emergence times that are muchquicker than secondary amplification reactions using universalamplification oligomers. In the linear multiplex pre-amplificationmethods, it is preferable, then that the secondary amplificationreactions use target specific amplification oligomers. Target specificamplification oligomers work well because the secondary reactions areseparated from each other, have no amplification oligomer carry overfrom the pre-amplification reaction, and do not have to contend withprimer interaction issues, and other similar problems common tomultiplex reactions.

Average emergence times for this example are as follows (n=4): PCA3 at 0copies/PSA at 1E3 copies average emergence time using target specificamplification oligomers was 18 minutes, average emergence time usinguniversal amplification oligomers was 52 minutes; PCA3 at 1E3 copies/PSAat 0 copies average emergence time using target specific amplificationoligomers was 15 minutes, average emergence time using universalamplification oligomers was 44 minutes; PCA3 at 1E5 copies/PSA at 1E3copies average emergence time for PSA using target specificamplification oligomers was 18 minutes, average emergence time for PSAusing universal amplification oligomers was 66 minutes, averageemergence time for PCA3 using target specific amplification oligomerswas 12 minutes, average emergence time for PSA using universalamplification oligomers was 32 minutes; and PCA3 at 1E3 copies/PSA at1E5 copies average emergence time for PSA using target specificamplification oligomers was 14 minutes, average emergence time for PSAusing universal amplification oligomers was 40 minutes, averageemergence time for PCA3 using target specific amplification oligomerswas 15 minutes, average emergence time for PSA using universalamplification oligomers was 44 minutes. Thus, in this example, theaverage emergence times were not affected by the presence of a 100-foldexcess of the other target in pre-amplification. To this point, PSA at1E3 copies showed an average emergence time of 18 minutes with orwithout 1E5 copies of competing PCA3 target and, similarly, PCA3 at 1E3copies showed an average emergence time of 15 minutes with or without1E5 copies of competing PSA target.

Example 14: Pre-Amplification Multiplex Reaction Using TSU AmplificationOligomers in the DH Format

This example is an 11-plex multiplex reaction wherein a target capturereagent comprises target capture, blocker and DH-complex oligomers forall 11 target nucleic acids. Following pre amplification, thepre-amplification product is split into eleven separate secondaryamplification reactions, each comprising target specific amplificationoligomers for one of the targets. Samples for this reaction comprisedvaried amounts of PCA3 target nucleic acid either alone (uniplex) orcombined with 8.75E5 copies of target nucleic acid from each of AMACR,CAP2, Chickengunyavirus (CHIKV), Erg exon 11, HIV pol, PCGEM1, PSA,PSGR, T2ERGc and West Nile Virus (WNV). Sample transport medium withoutadded target nucleic acids was used as the negative control. The targetcapture reagent comprised a DH-complex, a target capture oligomer and ablocker oligomer for each of the 11 target nucleic acids. It is notablethat CHIKV and WNV are forward TMA reactions, thus the T7 component ofthe DH-complex hybridizes to the target nucleic acid during the targetcapture step, whereas the remaining targets are reverse TMA and thenon-T7 hybridizes to those target nucleic acids during target capture.The member of the DH oligomer that does not initially hybridize thetarget nucleic acid during target capture is provided in an excessconcentration over the concentration of the member that does hybridize.For these examples, the DH oligomer member amounts are 7.5pmoles/reaction for the excess concentration and 5 pmoles/reaction forthe initially hybridizing member. Target capture was performed at60.deg.C for about 30 minutes followed by a room temperature incubationfor about 20 minutes. The captured target nucleic acids were then washedand eluted into a pre-amplification reaction mixture. Thepre-amplification reaction mixture comprised reverse transcriptase andRNA polymerase. The pre-amplification reaction was then incubated for 15minutes at 42.deg.C. Following pre-amplification, the pre-amplificationreaction is split into aliquots and then amplified in separate secondaryamplification reactions for one of 11 different targets, each comprisingtarget specific amplification oligomers and a molecular torch for one ofthe 11 target nucleic acids. The individual amplification reactions wereperformed at 42.deg.C for about 80 minutes and were monitored fordetection of amplification product throughout the amp reaction. Resultsfrom the PCA3 specific amplification is shown in Table 10. Each of theother target nucleic acids were successfully amplified in their owntarget specific secondary amplification reactions, data not shown.

TABLE 10 PCA3 PCA3 + Challenging Target PCA3 Uniplex Targets in 11-plexInput mean C (t) mean C(t) Copy # n = 4 % CV n = 4 % CV 100 21.5 4.07%20.9 4.84% 300 19.1 2.85% 19.6 3.03% 900 17.8 1.64% 18.0 1.32% 2,70016.6 1.25% 17.1 1.68% 8,100 16.0 0.64% 16.0 1.35% 24,300 15.0 0.44% 15.10.92% 72,900 14.1 0.46% 14.2 2.01% 218,700 13.4 1.08% 13.2 0.44%These results showed that PCA3 was amplified and detected following apre-amplification, and also when pre-amplified in the presence of alarge excess of 10 other target nucleic acids. Detection results forPCA3 in a uniplex reaction are similar to those for PCA3 in a multiplexreaction as shown in the table above. Amplification was also detectedfor these ten other target nucleic acids in this 11-plex reaction, withemergence times for 8.75E5 copies ranging from about 6 to about 15minutes of the amplification reaction.

In another part of this example, a calibration curve was constructedusing 10 fold dilutions of a calibrator nucleic acid (here, PCA3calibrator nucleic acid). The mean calculated log [copies] for eachtarget level were compared to known input log [copies]. The dependenceof these two values would ideally be expressed by equation y=x, where xis input log [copies] and y is calculated log [copies]. In this examplethe dependence was determined as: y=1.065x−0.090 with R2=0.9592 for PCA3alone, and y=1.0526x−0.084 with R2=0.99 for PCA3 in the presence ofchallenging targets. This shows a lack of interference of the PCA3pre-amplification and amplification reactions from other targets presentin the multiplex reaction.

Example 15: Alternate Designs for DH Linked TSU-Complexes

The DH linked amplification oligomer-complexes operate by hybridizingtogether the two oligomer members of the complex (e.g., the non-T7 to T7or the forward primer to reverse primer, etc). A plurality of variantamplification oligomer-complex oligomers members was prepared tocomprise a variety of complementary sequences for joining the T7 andNon-T7 members. These complementary sequences comprised all or part ofthe promoter sequence of the T7 member, and/or all or part of theuniversal tag sequence of the T7 member, and/or all or part of thetarget specific sequence of the T7 member. Amplificationoligomer-complex oligomer members targeting PSA, PCA3 and CAP2 wereprepared and all tested amplification oligomer-complex variants producedamplification products, though some were less robust than others,showing late emergence times and/or varied results for duplicatereactions. Nevertheless, these variant amplification oligomer-complexesare useful for amplification reactions. In addition to identifying thatthe variant amplification oligomer-complex oligomers worked inamplification reactions, a further discovery was made. Some of theseamplification oligomer-complex variants were resilient to unfavorableconditions such as temperature spikes, which cause the amplificationoligomer-complex members to dissociate.

In reactions wherein two or more DH-complexes are used, such as amultiplex reaction, dissociation of the oligomer members followed byreassociation at a lower temperature can result in mis-pairing. Thismeans that for a sample comprising amplification oligomer-complex A andamplification oligomer-complex B, wherein an event leads to adisassociation of the members of TSU complexes A & B, reassociation mayresult in the following amplification oligomer-complex species:amplification oligomer-complex A, amplification oligomer-complex B,amplification oligomer-complex AB & amplification oligomer-complex BA.Amplification oligomer-complexes AB & BA are mis-paired, and their usein an amplification reaction leads to inefficiencies in that reaction.One solution for preventing mis-pairing is the use of complementarypairing sequences that are unique to each species of amplificationoligomer-complex in the reaction system. Thus, when the dissociatedmembers of amplification oligomer-complex A and the dissociated membersof amplification oligomer-complex B reassociate, then the members of Aare more strongly driven together, and likewise, the members of B aremore strongly driven together. The following oligomers were designed toidentify unique pairing sequences for members of a TSU-complex targetingPCA3 target nucleic acid. Table 11. Though TSU-complexes areillustrated, these designs can also be applied to amplification oligomercomplexes lacking a universal tag sequence, wherein the complementaryportions of the oligomer members can include all or part of the promotersequence and all or part of the target specific sequence.

TABLE 11 SEQ ID Member NO: Sequence 5′→3′ Type  95aatttaatacgactcactatagggagaccacaacggttttaatgtctaagtagtg T7 ac  96tctccctatagtgagtcgtattaaattGTCATATGCGACGATCTCAGGGCTCATC Non-T7GATGACCCAAGATGGCGGC  97TCTCCCTATAGTGAGTCGGTCATATGCGACGATCTCAGGGCTCATCGATGACCCA Non-T7AGATGGCGGC  98 TCTCCCTATAGTGAGTCGGTCATATGCGACGATCTCAGGGCTCATCGATGACCCANon-T7 AGATGGCGGC  99TGGTCTCCCTATAGTGAGTCGGTCATATGCGACGATCTCAGGGCTCATCGATGAC Non-T7CCAAGATGGCGGC 100gtggtctccctatagtgagtcgGTCATATGCGACGATCTCAGGGCTCATCGATGA Non-T7CCCAAGATGGCGGC 101CCGTTGTGGTCTCCCTATAGTCATATGCGACGATCTCAGGGCTCATCGATGACCC Non-T7AAGATGGCGGC 102 CTTAGACGTGGTCTCCCTATAGTCATATGCGACGATCTCAGGGCTCATCGATGACNon-T7 CCAAGATGGCGGC 103CTTAGACATTTTGTGGTCTCCCGTCATATGCGACGATCTCAGGGCTCATCGATGA Non-T7CCCAAGATGGCGGC 104cttagaccgttgtggtctcccGTCATATGCGACGATCTCAGGGCTCATCGATGAC Non-T7CCAAGATGGCGGC 105ctacttagacatgtggtctcccGTCATATGCGACGATCTCAGGGCTCATCGATGA Non-T7CCCAAGATGGCGGC 106CACTACTTAGACAGGTCTCCCGTCATATGCGACGATCTCAGGGCTCATCGATGAC Non-T7CCAAGATGGCGGC 107CTTAGACATTAAAACCGTTGTGGGTCATATGCGACGATCTCAGGGCTCATCGATG Non-T7ACCCAAGATGGCGGC 108GTGGTCTCCCTATAGTGAGTCATATGCGACGATCTCAGGGCTCATCGATGACCCA Non-T7AGATGGCGGCSEQ ID NO:95 is the T7 member of the TSU-complexes and comprises apromoter sequence from residues 1-27, an universal sequence fromresidues 28-47 for subsequent amplification using an universalamplification oligomer and a target specific sequence. SEQ ID NOS:96-108are various non-T7 members of the TSU complexes and each comprise atarget specific sequence and an universal tag sequence at their 3′ ends(5′-GTCATATGCGACGATCTCAGGGCTCATCGATGACCCAAGATGGCGGC-3′). Joined to the 5prime end thereof are the varied DH complex forming sequences. SEQ IDNO:96 comprises a DH complex forming sequence that is the complement ofthe promoter sequence on the T7 member (cPRO). Thus, in a multiplexreaction all TSU complexes using the PRO-cPRO DH sequences would havethe same linking sequences, and could encounter thedissociation/reassociation problem mentioned above. Similarly, SEQ IDNOS:97-98 comprise a DH linking sequence that is complementary to aportion of the T7 promoter region, and could encounter the sameproblems. SEQ ID NOS:99-101 & 108 each comprise a DH linking sequencethat is complementary to a portion of the T7 promoter region and aportion of the universal tag sequence region. Because typically auniversal tag sequence is provided to allow for subsequent amplificationusing a single set of universal amplification oligomers, TSU complexesjoined by all or part of a PRO-cPRO sequence and/or all or part of auniversal tag-complement of universal tag sequence could encounter theassociation/dissociation problems discussed above. SEQ ID NOS:102-107comprise DH linking sequences that are complementary a portion of thePRO sequence and/or all or a part of the universal sequence, but alsoare partially complementary to the target specific portion of the T7member. Thus, each different TSU complex present in a multiplex reactioncould have unique DH linking sequences, thereby driving reassociationbetween the proper TSU complex members.

Each of the following combinations of TSU complexes were prepared (SEQID NOS:96 & 97; SEQ ID NOS:96 & 98; SEQ ID NOS:96 & 99; SEQ ID NOS:96 &100; SEQ ID NOS:96 & 101; SEQ ID NOS:96 & 102; SEQ ID NOS:96 & 103; SEQID NOS:96 & 104; SEQ ID NOS:96 & 105; SEQ ID NOS:96 & 106; SEQ ID NOS:96& 107; and SEQ ID NOS:96 & 108) and tested using PCA3 target nucleicacid. PCA3 samples were prepared to contain 2E2, 5E2, 1E3 or 5E3 copiesof target nucleic acids. Target capture reagents were prepared tocomprise a target capture oligomer, (SEQ ID NO:81), a blocker oligomer(SEQ ID NO:82) and one of the TSU complexes. Target capture wasperformed as is mentioned above and the captured targets from each ofthe capture reactions were resuspended into amplification reactionmixtures comprising universal amplification oligomers (SEQ ID NOS:93 &94) and a detection probe (SEQ ID NO:86). Amplification and detectionwere performed generally as is discussed herein. TSU complexescomprising SEQ ID NOS:95 & 103; and SEQ ID NOS:95 & 108 showed goodoverall performance and were selected for additional testing. Averageemergence times for these TSU complexes were as follows: for SEQ IDNOS:95 & 103 200 copies was 48.3 minutes, 500 copies was 49.2 minutes,1000 copies was 46.0 minutes and 5000 copies was 41.9 minutes; and forSEQ ID NOS:95 & 108 200 copies was 49.7 minutes, 500 copies was 47.0minutes, 1000 copies was 45.6 minutes and 5000 copies was 40.3 minutes.The TSU complexes comprising SEQ ID NOS:95 & 103; and SEQ ID NOS:95 &108 were then used in uniplex, duplex and triplex reactions wherein eachof the TSU-complexes in the reactions were subjected to dissociation andreassociation conditions.

For these uniplex, duplex and multiplex reactions, samples comprised 2E2copies or PSA, target nucleic acid, 2E2 copies of PCA3 target nucleicacid, 2E2 copies of CAP2 target nucleic acid, 1E3 copies of PSA targetnucleic acid, 1E3 copies of PCA3 target nucleic acid, 1E3 copies of CAP2target nucleic acid or combinations thereof, as is discussed in theresults section. Target capture reagents comprised target captureoligomer, blocker and one or more TSU complex, depending one whether thereaction was uniplex or multiplex, see Table 12.

TABLE 12 Target Nucleic Target Capture Blocker Acid(s) Oligomer(s)Oligomer(s) TSU Complex(s) 1. PSA Uniplex SEQ ID NO: 87 SEQ ID NO: 88SEQ ID NOS: 90 & 110 2. PSA Uniplex SEQ ID NO: 87 SEQ ID NO: 88 SEQ IDNOS: 90 & 109 3. PCA3 Uniplex SEQ ID NO: 87 SEQ ID NO: 82 SEQ ID NOS: 83& 84 4. PSA & PCA3 SEQ ID NOS: 81 & 87 SEQ ID NOS: 82 & 88 SEQ ID NOS:89, 90, Duplex 83 & 84 5. PSA & PCA3 SEQ ID NOS: 81 & 87 SEQ ID NOS: 82& 88 SEQ ID NOS: 90, 110, Duplex 95 & 108 6. PSA, PCA3 and SEQ ID NOS:75, 81 & SEQ ID NOS: 76, 82 & SEQ ID NOS: 77, 78, CAP2 Triplex 87 88 89,90, 83 & 84 7. PSA, PCA3 and SEQ ID NOS: 75, 81 & SEQ ID NOS: 76, 82 &SEQ ID NOS: 90, 110, CAP2 Triplex 87 88 95, 108, 78 & 112 8. PSA, PCA3and SEQ ID NOS: 75, 81 & SEQ ID NOS: 76, 82 & SEQ ID NOS: 90, 109, CAP2Triplex 87 88 95, 103, 78 & 111 9. PSA Uniplex SEQ ID NO: 87 SEQ ID NO:88 SEQ ID NOS: 89 & 90 10. PCA3 Uniplex SEQ ID NO: 81 SEQ ID NO: 82 SEQID NOS: 95 & 103 11. PSA, PCA3 and SEQ ID NOS: 75, 81 & SEQ ID NOS: 76,82 & SEQ ID NOS: 90, 109, CAP2 Triplex 87 88 78, 111, 95 & 103Each of the target capture reagents in Table 12 were made in duplicate,one member of which was subjected to a 72.deg.C temperature spike beforeuse, while the other of which was stored at a consistent 25.deg.C untilused. The temperature spike was applied to one of the samples to createa dissociation/reassociation condition. Target capture was thenperformed generally as is described herein. Samples were resuspendedinto amplification reactions comprising universal amplificationoligomers (SEQ ID NOS:93 & 94) and molecular torches for real timedetection of amplification product (for amplifications wherein PSAtarget nucleic acids were included, SEQ ID NO:92; for amplificationswherein PCA3 target nucleic acids were included, SEQ ID NO:86; foramplifications wherein CAP2 target nucleic acids were included, SEQ IDNO:80). Amplifications and real-time detections were performed generallyas is described herein.

The TSU complexes in target capture reagents 3, 4, 6 & 9 from Table 12comprise DH linkages wherein a complementary promoter sequence on thenon-T7 amplification oligomer member hybridizes to the promoter sequenceon the T7 oligomer member. The TSU complexes in target capture reagents1, 5 & 7 from Table 12 comprise DH linkages wherein the Non-T7 linkagesequences are complementary to part of the promoter sequence and part ofthe universal tag sequence of the T7 member. The TSU complexes in targetcapture reagents 2, 8, 10 & 11 from Table 12 comprise DH linkageswherein the Non-T7 linkage sequences are complementary to part of thepromoter sequence, part of the universal tag sequence and part of thetarget specific sequence of the T7 member. In a first set ofexperiments, target capture reagents 4, 6 & 9 were tested forperformance following two sets of conditions; the first set ofconditions held the TSU complexes at 25.deg.C until used for a targetcapture reaction while the second set of conditions provided a 72.deg.temperature spike before use. Results showed that when the TSU complexwas held at a about 25.deg.C, the uniplex, duplex and triplex reactionsall provided very similar amplification results (Table 13). However,when the TSU oligomers experienced a 72.deg.C temperature spike beforebeing used in the target capture procedure, the amplification resultswere not as good. Here, though the uniplex reaction results were similarto those for the consistent temperature reactions (Table 13), the duplexand triplex reactions were delayed and less robust (Table 13). In asecond set of experiments, target capture reagents 1, 2, 5, 7 & 8 weretested for performance following a 72.deg.C temperature spike. The TSUcomplexes used for this second experiment showed improvement over theabove temperature spike results for the duplex and triplex reactions.The triplex reactions using the TSU complexes in target capture reactionmix 7 (triplex oligomer) were slower and less robust than those intarget capture reaction mixtures 1 (uniplex) and 5 (duplex) (Table 13).Target capture reaction mixes 2 & 8 showed very good and very consistentresults for all reactions (Table 13). TSU-complexes joined by uniqueDH-linkage sequences, such as using at least a portion of the targetsequence of one member of the TSU complex, perform well following adissociation and reassociation compared to those that are linked usingsequences that are common or universal amongst the species ofTSU-complex in a mix.

TABLE 13 Emergence time (minutes) 200 copies per 1,000 copies per TCRreaction reaction Plex # Room temp 72-deg-C. Room temp 72-deg-C. AUniplex 9 63 65 58 60 Duplex 4 63 67 58 64 Triplex 6 63 70 58 62 BUniplex 1 — 63 — 57 Duplex 5 — 63 — 58 Triplex 7 — 63 — 63 C Uniplex 2 —62 — 56 Triplex 8 — 62 — 57

Example 16: Alternate Designs for TSU-Complexes

The TSU-complexes can comprise a variety of different amplificationoligomer types. In one example, the amplification oligomer members couldcomprise forward and reverse primer oligomers. These oligomers could belinked using a direct linkage format, such as covalently linking eachmember to the other. The primer members forming the complex would beoriented to each provide their 3′ end for a nucleic acid extensionreaction. The primer members could further and optionally compriseuniversal sequences, which would allow for secondary amplification usinguniversal amplification oligomers. These TSU primer complexes could behybridized to a target nucleic acid, and a first extension reactioncould be performed therefrom to generate a double stranded initialamplification product comprising the sequences of these TSU-complexmembers. Secondary amplification could take place therefrom usinguniversal amplification oligomers. PCR amplification and otheramplification methods are well known in the art.

In one example of using the TSU complex, a multiplex reaction could beperformed. In such a reaction, a sample suspected of comprising two ormore target nucleic acids of interest could be provided. Exemplarytarget nucleic acids could include, but are not limited to, thosewherein the target nucleic acids are part of a larger nucleic acid, suchas different target sequence regions of a mitochondrial DNA, ordifferent target sequences of an HLA nucleic acid; or those that areseparate, but jointly interesting target nucleic acids, such as two ormore species of Mycoplasma from a bioreactor, two or more potentiallyinfectious bacterium from a hospital setting, and the like. TSUcomplexes configured to hybridize with the target nucleic acidssuspected to be in the sample are then added into the sample. A targetcapture system may be included as well. Exemplary, but non-limiting,target capture systems are discussed herein. A first amplificationreaction, e.g., a PCR amplification reaction, is then performed usingthese TSU complexes in order to integrate the TSU complex membersequences. Subsequent amplification reactions, e.g., PCR, could then beperformed using, for example, universal amplification oligomers. Targetspecific detection could then be performed. One method of targetspecific amplification product detection is the use of molecular probes.Others include mass spectrometry, nucleic acid sequencing and gelelectrophoresis.

The invention claimed is:
 1. A target capture reaction mixture, whereinthe reaction mixture comprises at least one target nucleic acid, atleast one target capture oligonucleotide and at least one amplificationoligomer complex, wherein each of said amplification oligomer complexescomprises a first amplification oligomer member having a first targetspecific sequence that is joined to a second amplification oligomermember having a second target specific sequence, wherein the firstamplification oligomer member is a non-promoter primer and the secondamplification oligomer member is a promoter primer, and wherein each ofsaid target capture oligomers and each of said amplification oligomercomplexes specifically hybridizes to a different target nucleic acidsequence.
 2. The target capture reaction mixture of claim 1, furthercomprising at least one solid support.
 3. The target capture reactionmixture of claim 2, wherein said solid support is a magbead.
 4. Thetarget capture reaction mixture of claim 1, wherein said at least onetarget capture oligomer is a wobble probe.
 5. The target capturereaction mixture of claim 1, further comprising from about 0.05M toabout 4.2M of an imidazolium compound.
 6. The target capture reactionmixture of claim 1, wherein at least one of the amplification oligomercomplexes is a DH-complex.
 7. The target capture reaction mixture ofclaim 6, wherein the first amplification oligomer member of at least oneof the amplification oligomer complexes is joined on its 5′ end to alinking member for linking the first amplification oligomer member tothe second amplification oligomer member of the amplification oligomercomplex.
 8. The target capture reaction mixture of claim 7, wherein thelinking member is a nucleotide sequence that is complementary to aportion of a nucleotide sequence of the second amplification oligomermember.
 9. The target capture reaction mixture of claim 8, wherein thelinking member is a nucleotide sequence that is complementary to apromoter sequence of the second amplification oligomer member.
 10. Thetarget capture reaction mixture of claim 1, wherein the secondamplification oligomer member comprises a blocked 3′ terminus.
 11. Thetarget capture reaction mixture of claim 1, wherein the firstamplification oligomer member of at least one of the amplificationoligomer complexes is joined on its 5′ end to a linking member forlinking the first amplification oligomer member to the secondamplification oligomer member of the amplification oligomer complex. 12.The target capture reaction mixture of claim 11, wherein the linkingmember is a nucleotide sequence that is complementary to a portion of anucleotide sequence of the second amplification oligomer member.
 13. Thetarget capture reaction mixture of claim 12, wherein the linking memberis a nucleotide sequence that is complementary to a promoter sequence ofthe second amplification oligomer member.
 14. The target capturereaction mixture of claim 11, further comprising from about 0.05M toabout 4.2M of an imidazolium compound.
 15. A target capture reactionmixture, wherein the reaction mixture comprises (a) at least one targetnucleic acid, (b) at least one target capture oligonucleotide, (c) atleast one solid support, (d) at least one amplification oligomercomplex, wherein each of said amplification oligomer complexes comprisesa first amplification oligomer member having a first target specificsequence that is joined to a second amplification oligomer member havinga second target specific sequence, wherein the first amplificationoligomer member is a non-promoter primer and the second amplificationoligomer member is a promoter primer, wherein the first amplificationoligomer member is joined on its 5′ end to a linking member for linkingthe first amplification oligomer member to the second amplificationoligomer member, wherein each of said target capture oligomers and eachof said amplification oligomer complexes specifically hybridizes to adifferent target nucleic acid sequence, and wherein each target nucleicacid sequence is specifically targeted by one of said target captureoligomers and one of said amplification oligomer complexes.
 16. Thetarget capture reaction mixture of claim 15, wherein the linking memberis a nucleotide sequence that is complementary to a portion of anucleotide sequence of the second amplification oligomer member.
 17. Thepre-amplification reaction mixture of claim 16, wherein the linkingmember is a nucleotide sequence that is complementary to a promotersequence of the second amplification oligomer member.
 18. A targetcapture reaction mixture, wherein the reaction mixture comprises (a) atleast one target nucleic acid, (b) at least one target captureoligonucleotide, (c) at least one solid support, wherein said solidsupport is a magbead, (d) at least one amplification oligomer complex,wherein each of said amplification oligomer complexes comprises a firstamplification oligomer member having a first target specific sequencethat is joined to a second amplification oligomer member having a secondtarget specific sequence, wherein the first amplification oligomermember is a non-promoter primer and the second amplification oligomermember is a promoter primer, wherein the first amplification oligomermember is joined on its 5′ end to a linking member that is a nucleotidesequence that is substantially complementary to the promoter sequence ofthe second amplification oligomer for linking the first amplificationoligomer member to the second amplification oligomer member, whereineach of said target capture oligomers and each of said amplificationoligomer complexes specifically hybridizes to a different target nucleicacid sequence, and wherein each target nucleic acid sequence isspecifically targeted by one of said target capture oligomers and one ofsaid amplification oligomer complexes.
 19. The target capture reactionmixture of claim 18, wherein the second amplification oligomer membercomprises a blocked 3′ terminus.
 20. The target capture reaction mixtureof claim 18, further comprising from about 0.05M to about 4.2M of animidazolium compound.